Methods and compositions for identifying receptor effectors

ABSTRACT

The present invention makes available a rapid, effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a cellular receptor or ion channel. The subject assay enables rapid screening of large numbers of polypeptides in a library to identifying those polypeptides which induce or antagonize receptor bioactivity. The subject assay is particularly amenable for identifying surrogate ligands for orphan receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 08/582,333filed on Jan. 17, 1996 (now allowed), which in turn is acontinuation-in-part application of Ser. No. 08/464,531, filed Jun. 5,1995 (now issued as U.S. Pat. No. 5,789,184,), which in turn is acontinuation-in-part application of Ser. No. 08/461,598, filed Jun. 5,1995 (now issued as U.S. Pat. No. 5,876,951), which in turn is acontinuation-in-part application of Ser. No. 08/461,383, filed on Jun.5, 1995 (now abandoned), which in turn is a continuation-in-partapplication of Ser. No. 08/463,181 filed on Jun. 5, 1995 (nowabandoned), which in turn is a continuation-in-part application of Ser.No. 08/322,137 filed on Oct. 13, 1994 (now issued as U.S. Pat. No.6,100,042), which in turn is a continuation-in-part application of Ser.No. 08/309,313 filed on Sep. 20, 1994 (now abandoned), which in turn isa continuation-in-part application of Ser. No. 08/190,328 filed on Jan.31, 1994 (now abandoned), which in turn is a continuation-in-partapplication of Ser. No. 08/041,431 filed on Mar. 31, 1993 (nowabandoned).

BACKGROUND OF THE INVENTION

A common technique for cloning receptors is to use nucleic acidhybridization technology to identify receptors which are homologous toother, known receptors. For instance, originally the cloning of seventransmembrane domain G protein-coupled receptors (GCR) depended on theisolation and sequencing of the corresponding protein or the use ofexpression cloning techniques. However, when sequences for thesereceptors became available, it was apparent that there were significantsequence homologies between these receptors. This technology, since itdoes not require that the ligand of the receptor have been identified,has resulted in the cloning of a large number of “orphan receptors”,which have no known ligand and often whose biological function isobscure. Receptors of all types comprise this large family. Known orphanreceptors include the nuclear receptors COUP-TF1/EAR3, COUP-TF2/ARP1,EAR-1, EAR-2, TR-2, PPAR1, HNF-4, ERR-1, ERR-2, NGFIB/Nur77, ELP/SF-1and MPL (Parker et al, supra, and Power et al. (1992) TIBS 13:318–323).A large number of orphan receptors have been identified in the EPHfamily (Hirai et al (1987) Science 238:1717–1720). HER3 and HER4 areorphan receptors in the epidermal growth factor receptor family (Plowmanet al. (1993) Proc. Natl. Acad. Sci. USA 90:1746–1750). ILA is a newlyidentified member of the human nerve growth factor/tumor necrosis factorreceptor family (Schwarz et al. (1993) Gene 134:295–298). IRRR is anorphan insulin receptor-related receptor which is a transmembranetyrosine kinase (Shier et al. (1989) J. Biol Chem 264:14606–14608).Several orphan tyrosine kinase receptors have been found in Drosophila(Perrimon (1994) Curr. Opin. Cell Biol. 6:260–266). The importance ofidentifying ligands for orphan receptors is clear; it opens up a widearea for research in the area of drug discovery.

One large subgroup of orphan receptors, as alluded to above, are foundin the G protein coupled receptor family. Approximately 100 suchreceptors have been identified by function and these mediatetransmembrane signaling from external stimuli (vision, taste and smell),endocrine function (pituitary and adrenal), exocrine function(pancreas), heart rate, lipolysis, and carbohydrate metabolism.Structural and genetic similarities suggest that G protein-coupledreceptor superfamily can be subclassified into five distinct groups: (i)amine receptors (serotonin, adrenergic, etc.); (ii) small peptidehormone (somatostatin, TRH, etc.); (iii) large peptide hormone (LH-CG,FSH, etc.); (iv) secretin family; and (v) odorant receptors (Buck L. andAxel, R. (1991) Cell 65:175–187), with orphan receptors apparentlyoccurring in each of the sub-families.

Previous work describes the expression of recombinant mammalian Gprotein-coupled receptors as a means of studying receptor function as ameans of identifying agonists and antagonists of those receptors. Forexample, the human muscarinic receptor (HM1) has been functionallyexpressed in mouse cells (Harpold et al. U.S. Pat. No. 5,401,629). Therat V1b vasopressin receptor has been found to stimulatephosphotidy.inositol hydrolysis and intracellular Ca2+ mobilization inChinese hamster ovary cells upon agonist stimulation (Lolait et al.(1995) Proc Natl. Acad Sci. USA 92:6783–6787). Likewise, the C5areceptor {to be completed} These types of ectopic expression studieshave enabled researchers to study receptor signalling mechanisms and toperform mutagenisis studies which have been useful in identifyingportions of receptors that are critical for ligand binding or signaltransduction.

Experiments have also been undertaken to express functional G proteincoupled receptors in yeast cells. For example, U.S. Pat. No. 5,482,835to King et al. describes a transformed yeast cell which is incapable ofproducing a yeast G protein α subunit, but which has been engineered toproduce both a mammalian G protein α-subunit and a mammalian receptorwhich is “coupled to” (i.e., interacts with) the aforementionedmammalian G protein α-subunit. Specifically, U.S. Pat. No. 5,482,835reports expression of the human beta-2 adrenergic receptor (β2AR), aseven transmembrane receptor (STR), in yeast, under control of the GAL1promoter, with the β2AR gene modified by replacing the first 63 basepairs of coding sequence with 11 base pairs of noncoding and 42 basepairs of coding sequence from the STE2 gene. (STE2 encodes the yeastα-factor receptor). The Duke researchers found that the modified β2ARwas functionally integrated into the membrane, as shown by studies ofthe ability of isolated membranes to interact properly with variousknown agonists and antagonists of β2AR. The ligand binding affinity foryeast-expressed β2AR was said to be nearly identical to that observedfor naturally produced β2AR.

U.S. Pat. No. 5,482,835 describes co-expression of a rat G proteinα-subunit in the same cells, yeast strain 8C, which lacks the cognateyeast protein. Ligand binding resulted in G protein-mediated signaltransduction. U.S. Pat. No. 5,482,835 teaches that these cells may beused in screening compounds for the ability to affect the rate ofdissociation of Gα from Gβγ in a cell. For this purpose, the cellfurther contains a pheromone-responsive promoter (e.g. BAR1 or FUS1),linked to an indicator gene (e.g. HIS3 or LacZ). The cells are placed inmulti-titer plates, and different compounds are placed in each well. Thecolonies are then scored for expression of the indicator gene.

SUMMARY OF THE INVENTION

The present invention relates to a rapid, reliable and effective assayfor screening and identifying pharmaceutically effective compounds thatspecifically interact with and modulate the activity of a cellularreceptor or ion channel. The subject assay enables rapid screening oflarge numbers of polypeptides in a library to identifying thosepolypeptides which agonize or antagonize receptor bioactivity. Ingeneral, the assay is characterized by the use of a library ofrecombinant cells, each cell of which include (i) a target receptorprotein whose signal transduction activity can be modulated byinteraction with an extracellular signal, the transduction activitybeing able to generate a detectable signal, and (ii) an expressiblerecombinant gene encoding an exogenous test polypeptide from apolypeptide library. By the use of a variegated gene library, themixture of cells collectively express a variegated population of testpolypeptides. In preferred embodiments, the polypeptide library includesat least 10³ different polypeptides, though more preferably at least10⁵, 10⁶, or 10⁷ different (variegated) polypeptides. The polypeptidelibrary can be generated as a random peptide library, as a semi-randompeptide library (e.g., based on combinatorial mutagenesis of a knownligand), or as a cDNA library.

The ability of particular constituents of the peptide library tomodulate the signal transduction activity of the target receptor can bescored for by detecting up or down-regulation of the detection signal.For example, second messenger generation via the receptor can bemeasured directly. Alternatively, the use of a reporter gene can providea convenient readout. In any event, a statistically significant changein the detection signal can be used to facilitate isolation of thosecells from the mixture which contain a nucleic acid encoding a testpolypeptide which is an effector of the target receptor.

By this method, test polypeptides which induce receptor signaling can beidentified. If the test polypeptide does not appear to directly inducethe activity of the receptor protein, the assay may be repeated andmodified by the introduction of a step in which the recombinant cell isfirst contacted with a known activator of the target receptor to inducethe signal transdution pathways from the receptor. In one embodiment,the test polypeptide is assayed for its ability to antagonize, e.g.,inhibit or block the activity of the activator. Alternatively, the assaycan score for peptides from the peptide library which potentiate theinduction response generated by treatment of the cell with a knownactivator. As used herein, an “agonist” refers to agents which eitherinduce activation of receptor signalling pathways, e.g., such as bymimicking a ligand for the receptor, as well as agents which potentiatethe sensitivity of the receptor to a ligand, e.g., lower theconcentrations of ligand required to induce a particular level ofreceptor-dependent signalling.

In one embodiment of the present invention the reagent cells express thereceptor of interest endogenously. In yet other embodiments, the cellsare engineered to express a heterlogous target receptor protein. Ineither of these embodiments, it may be desirable to inactivate one ormore endogenous genes of the host cells. For example, certain preferredembodiments in which a heterlogous receptor is provided utilize hostcells in which the gene for the homologous receptor has beeninactivated. Likewise, other proteins involved in transducing signalsfrom the target receptor can be inactivated, or complemented with anortholog or paralog from another organism, e.g., yeast G proteinsubunits can be complemented by mammalian G protein subunits in yeastcells also engineered to express a mammalian G protein coupled receptor.Other complementations include, for example, expression of heterologousMAP kinases or erk kinases, MEKs or MKKs (MAP kinase kinases), MEKKs(MEK kinases), ras, raf, STATs, JAKs and the like.

The receptor protein can be any receptor which interacts with anextracellular molecule (i.e. hormone, growth factor, peptide) tomodulate a signal in the cell. To illustrate the receptor can be a cellsurface receptor, or in other embodiments can be an intracellularreceptor. In preferred embodiments, the receptor is a cell surfacereceptor, such as: a receptor tyrosine kinase, e.g., an EPH receptor; anion channel; a cytokine receptor; an multisubunit immune recognitionreceptor, a chemokine receptor; a growth factor receptor, or a G-proteincoupled receptor, such as a chemoattracttractant peptide receptor, aneuropeptide receptor, a light receptor, a neurotransmitter receptor, ora polypeptide hormone receptor.

Preferred G protein coupled receptors include α1A-adrenergic receptor,α1B-adrenergic receptor, α2-adrenergic receptor, α2B-adrenergicreceptor, β1-adrenergic receptor, β2-adrenergic receptor, β3-adrenergicreceptor, m1 acetylcholine receptor (AChR), m2 AChR, m3 AChR, m4 AChR,m5 AChR, D1 dopamine receptor, D2 dopamine receptor, D3 dopaminereceptor, D4 dopamine receptor, D5 dopamine receptor, A1 adenosinereceptor, A2b adenosine receptor, 5-HT1a receptor, 5-HT1b receptor,5HT1-like receptor, 5-HT1d receptor, 5HT1d-like receptor, 5HT1d betareceptor, substance K (neurokinin A) receptor, fMLP receptor, fMLP-likereceptor, angiotensin II type 1 receptor, endothelin ETA receptor,endothelin ETB receptor, thrombin receptor, growth hormone-releasinghormone (GHRH) receptor, vasoactive intestinal peptide receptor,oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3, cannabinoidreceptor, follicle stimulating hormone (FSH) receptor, leutropin(LH/HCG) receptor, thyroid stimulating hormone (TSH) receptor,thromboxane A2 receptor, platelet-activating factor (PAF) receptor, C5aanaphylatoxin receptor, Interleukin 8 (IL-8) IL-8RA, IL-8RB, DeltaOpioid receptor, Kappa Opioid receptor, mip-1/RANTES receptor,Rhodopsin, Red opsin, Green opsin, Blue opsin, metabotropic glutamatemGluR1–6, histamine H2 receptor, ATP receptor, neuropeptide Y receptor,amyloid protein precursor receptor, insulin-like growth factor IIreceptor, bradykinin receptor, gonadotropin-releasing hormone receptor,cholecystokinin receptor, melanocyte stimulating hormone receptorreceptor, antidiuretic hormone receptor, glucagon receptor, andadrenocorticotropic hormone II receptor.

Preferred EPH receptors include eph, elk, eck, sek, mek4, hek, hek2,eek, erk, tyrol, tyro4, tyro5, tyro6, tyro11, cek4, cek5, cek6, cek7,cek8, cek9, cek10, bsk, rtk1, rtk2, rtk3, myk1, myk2, ehk1, ehk2,pagliaccio, htk, erk and nuk receptors.

As set forth below, no matter which structural/function class to whichthe target receptor may belong, the subject assay is amenable toidentifying ligands for an otherwise orphan receptor.

In those embodiments wherein the target receptor is a cell surfacereceptor, it will be desirable for the peptides in the library toexpress a signal sequence to ensure that they are processed in theappropriate secretory pathway and thus are available to interact withreceptors on the cell surface.

With respect to a detection signal generated by signal transduction,certain of the preferred embodiments measure the production of secondmessengers to determine changes in ligand engagement by the receptor. Inpreferred embodiments, changes in GTP hydrolysis, calcium mobilization,or phospholipid hydrolysis can be measured.

In other preferred embodiment, the host cells harbors a reporterconstruct containing a reporter gene in operative linkage with one ormore transcriptional regulatory elements responsive to the signaltransductin activity of the receptor protein. Exemplary reporter genesinclude enzymes, such as luciferase, phosphatase, or β-galactosidasewhich can produce a spectrometrically active label, e.g., changes incolor, fluorescence or luminescence, or a gene product which alters acellular phenotype, e.g., cell growth, drug resistance or auxotrophy. Inpreferred embodiments: the reporter gene encodes a gene product selectedfrom the group consisting of chloramphenicol acetyl transferase,beta-galactosidase and secreted alkaline phosphatase; the reporter geneencodes a gene product which confers a growth signal; the reporter geneencodes a gene product for growth in media containing aminotriazole orcanavanine.

The reagent cells of the present invention can be derived from anyeukaryotic organism. In preferred embodiments the cells are mammaliancells. In more preferred embodiments the cells are yeast cells, withcells from the genera Saccharomyces or Schizosaccharomyces being morepreferred. However, cells from amphibia (such as xenopus), avian orinsect sources are also contemplated. The host cells can derived fromprimary cells, or transformed and/or immortalized cell lines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures of pAAH5 and pRS-ADC.

FIG. 2. Schematic diagram of the structure of the plasmid resulting frominsertion of random oligonucleotides into pADC-MF alpha. This plasmidexpresses random peptides in the context of the MF alpha 1 signal andleader peptide.

FIG. 3. Schematic diagram of the structure of the plasmid resulting frominsertion of random oligonucleotides into pADC-MFa. This plasmidexpresses random peptides in the context of the MFa1 leader andC-terminal CVIA tetrapeptide.

FIG. 4. Activity of a fus1 promoter in response to signaling by humanC5a expressed in autocrine strains of yeast.

DETAILED DESCRIPTION OF THE INVENTION

Proliferation, differentiation and death of eukaryotic cells arecontrolled by hormones, neurotransmitters, and polypeptide factors.These diffusible ligands allow cells to influence and be influenced byenvironmental cues. The study of receptor-ligand interaction hasrevealed a great deal of information about how cells respond to externalstimuli, and this knowledge has led to the development oftherapeutically important compounds. However, the rate at whichreceptors have been cloned has recently increased dramatically—existingfamilies have been extended and new families recognized. In particular,the application of advanced cloning approaches has allowed the isolationof many receptors for which ligands are initially unknown. These arecommonly referred to in the art as “orphan” receptors, and several havesubsequently proved to be important pharmacological targets.

The present invention makes available a rapid, effective assay forscreening and identifying pharmaceutically effective compounds thatspecifically interact with and modulate the activity of a cellularreceptor or ion channel. The subject assay enables rapid screening oflarge numbers of polypeptides in a library to identifying thosepolypeptides which induce or antagonize receptor bioactivity.

In general, the assay is characterized by the use of a mixture ofrecombinant cells to sample a variegated polypeptide library forreceptor agonists or antagonists. As described with greater detailbelow, the reagent cells express both a target receptor protein capableof transducing a detectable signal in the reagent cell, and a testpolypeptide for which interaction with the receptor is to beascertained. Collectively, a culture of such reagent cells will providea variegated library of potential receptor effectors and those membersof the library which either agonize or antagonize the receptor functioncan be selected and identified by sequence.

One salient feature of the subject assay is the enhanced sensitivityresulting from expression of the test polypeptide in a cell which alsoserves as a reporter for the desired receptor-ligand interaction. Toillustrate, where the detectable signal resulting from receptorengagement by an agonist provides a growth signal or drug resistance,individual cells expressing polypeptides which agonize receptor functioncan be amplified and isolated from a library culture.

Accordingly, the present invention provides a convenient format fordiscovering drugs which can be useful to modulate cellular function, aswell as to understand the pharmacology of compounds that specificallyinteract with cellular receptors or ion channels. Moreover, the subjectassay is particularly amenable to identifying ligands, natural orartifical, for orphan receptors.

Before further description of the invention, certain terms employed inthe specification, examples and appended claims are, for convenience,collected here.

As used herein, “recombinant cells” include any cells that have beenmodified by the introduction of heterologous DNA. Control cells includecells that are substantially identical to the recombinant cells, but donot express one or more of the proteins encoded by the heterologous DNA,e.g., do not include or express the reporter gene construct, receptor ortest polypeptide.

The terms “recombinant protein”, “heterologous protein” and “exogenousprotein” are used interchangeably throughout the specification and referto a polypeptide which is produced by recombinant DNA techniques,wherein generally, DNA encoding the polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. That is, the polypeptide isexpressed from a heterologous nucleic acid.

As used herein, “heterologous DNA” or “heterologous nucleic acid”include DNA that does not occur naturally as part of the genome in whichit is present or which is found in a location or locations in the genomethat differs from that in which it occurs in nature. Heterologous DNA isnot endogenous to the cell into which it is introduced, but has beenobtained from another cell. Generally, although not necessarily, suchDNA encodes RNA and proteins that are not normally produced by the cellin which it is expressed. Heterologous DNA may also be referred to asforeign DNA. Any DNA that one of skill in the art would recognize orconsider as heterologous or foreign to the cell in which is expressed isherein encompassed by heterologous DNA. Examples of heterologous DNAinclude, but are not limited to, DNA that encodes test polypeptides,receptors, reporter genes, transcriptional and translational regulatorysequences, selectable or traceable marker proteins, such as a proteinthat confers drug resistance.

As used herein, “cell surface receptor” refers to molecules that occuron the surface of cells, interact with the extracellular environment,and transmit or transduce the information regarding the environmentintracellularly in a manner that ultimately modulates transcription ofspecific promoters, resulting in transcription of specific genes.

As used herein, “extracellular signals” include a molecule or a changein the environment that is transduced intracellularly via cell surfaceproteins that interact, directly or indirectly, with the signal. Anextracellular signal or effector molecule includes any compound orsubstance that in some manner specifically alters the activity of a cellsurface protein. Examples of such signals include, but are not limitedto, molecules such as acetylcholine, growth factors and hormones, thatbind to cell surface and/or intracellular receptors and ion channels andmodulate the activity of such receptors and channels.

As used herein, “extracellular signals” also include as yet unidentifiedsubstances that modulate the activity of a cellular receptor, andthereby influence intracellular functions. Such extracellular signalsare potential pharmacological agents that may be used to treat specificdiseases by modulating the activity of specific cell surface receptors.

“Orphan receptors” is a designation given to a receptors for which nospecific natural ligand has been described.

As used herein, a “reporter gene construct” is a nucleic acid thatincludes a “reporter gene” operatively linked to a transcriptionalregulatory sequences. Transcription of the reporter gene is controlledby these sequences. The activity of at least one or more of thesecontrol sequences is directly or indirectly regulated by the targetreceptor protein. The transcriptional regulatory sequences include thepromoter and other regulatory regions, such as enhancer sequences, thatmodulate the activity of the promoter, or regulatory sequences thatmodulate the activity or efficiency of the RNA polymerase thatrecognizes the promoter, or regulatory sequences are recognized byeffector molecules, including those that are specifically induced byinteraction of an extracellular signal with the target receptor. Forexample, modulation of the activity of the promoter may be effected byaltering the RNA polymerase binding to the promoter region, or,alternatively, by interfering with initiation of transcription orelongation of the mRNA. Such sequences are herein collectively referredto as transcriptional regulatory elements or sequences. In addition, theconstruct may include sequences of nucleotides that alter translation ofthe resulting mRNA, thereby altering the amount of reporter geneproduct.

“Signal transduction” is the processing of chemical signals from thecellular environment through the cell membrane, and may occur throughone or more of several mechanisms, such as phosphorylation, activationof ion channels, effector enzyme activation via guanine nucleotidebinding protein intermediates, formation of inositol phosphate,activation of adenylyl cyclase, and/or direct activation (or inhibition)of a transcriptional factor.

The term “modulation of a signal transduction activity of a receptorprotein” in its various grammatical forms, as used herein, designatesinduction and/or potentiation, as well as inhibition of one or moresignal transduction pathways downstream of a receptor.

Agonists and antagonists are “receptor effector” molecules that modulatesignal transduction via a receptor. Receptor effector molecules arecapable of binding to the receptor, though not necessarily at thebinding site of the natural ligand. Receptor effectors can modulatesignal transduction when used alone, i.e. can be surrogate ligands, orcan alter signal transduction in the presence of the natural ligand,either to enhance or inhibit signaling by the natural ligand. Forexample, “antagonists” are molecules that block or decrease the signaltransduction activity of receptor, e.g., they can competitively,noncompetitively, and/or allosterically inhibit signal transduction fromthe receptor, whereas “agonists” potentiate, induce or otherwise enhancethe signal transduction activity of a receptor. The terms “receptoractivator” and “surrogate ligand” refer to an agonist which inducessignal transduction from a receptor.

The term “substantially homologous”, when used in connection with aminoacid sequences, refers to sequences which are substantially identical toor similar in sequence, giving rise to a homology in conformation andthus to similar biological activity. The term is not intended to imply acommon evolution of the sequences.

Typically, “substantially homologous” sequences are at least 50%, morepreferably at least 80%, identical in sequence, at least over anyregions known to be involved in the desired activity. Most preferably,no more than five residues, other than at the termini, are different.Preferably, the divergence in sequence, at least in the aforementionedregions, is in the form of “conservative modifications”.

The term “autocrine cell”, as used herein, refers to a cell whichproduces a substance which can stimulate a receptor located on or withinthe same cell as produces the substance. For example, wild-type yeast αand a cells are not autocrine. However, a yeast cell which produces bothα-factor and α-factor receptor, or both α-factor and α-factor receptor,in functional form, is autocrine. By extension, cells which produce apeptide which is being screened for the ability to activate a receptor(e.g., by activating a G protein-coupled receptor) express the receptorare called “autocrine cells”, though it might be more precise to callthem “putative autocrine cells”. Of course, in a library of such cells,in which a multitude of different peptides are produced, it is likelythat one or more of the cells will be “autocrine” in the stricter senseof the term.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein.

I. Overview of Assay

As set out above, the present invention relates to methods foridentifying effectors of a receptor protein or complex thereof. Ingeneral, the assay is characterized by the use of a library ofrecombinant cells, each cell of which include (i) a target receptorprotein whose signal transduction activity can be modulated byinteraction with an extracellular signal, the transduction activitybeing able to generate a detectable signal, and (ii) an expressiblerecombinant gene encoding an exogenous test polypeptide from apolypeptide library. By the use of a variegated gene library, themixture of cells collectively express a variegated population of testpolypeptides.

The ability of particular constituents of the peptide library tomodulate the signal transduction activity of the target receptor can bescored for by detecting up or down-regulation of the detection signal.For example, second messenger generation (e.g. GTPase activity,phospholipid hydrolysis, or protein phosphorylation) via the receptorcan be measured directly. Alternatively, the use of a reporter gene canprovide a convenient readout. In any event, a statistically significantchange in the detection signal can be used to facilitate isolation ofthose cells from the mixture which contain a nucleic acid encoding atest polypeptide which is an effector of the target receptor.

By this method, test polypeptides which induce the receptor's signalingcan be screened. If the test polypeptide does not appear to induce theactivity of the receptor protein, the assay may be repeated and modifiedby the introduction of a step in which the recombinant cell is firstcontacted with a known activator of the target receptor to induce signaltransduction from the receptor, and the test polypeptide is assayed forits ability to inhibit the activity of the receptor, e.g., to identifyreceptor antagonists. In yet other embodiments, the peptide library canbe screened for members which potentiate the response to a knownactivator of the receptor. In this respect, surrogate ligands identifiedby the present assay for orphan receptors can be used as the exogenousactivator, and further peptide libraries screened for members whichpotentiate or inhibit the activating peptide. Alternatively, thesurrogate ligand can be used to screen exogenous compound libraries(peptide and non-peptide) which, by modulating the activity of theidentified surrogate, will presumably also similarly effect the nativeligand's effect on the target receptor. In such embodiments, thesurrogate ligand can be applied to the cells, though is preferablyproduced by the reagent cell, thereby providing an autocrine cell.

In developing the recombinant cells assays, it was recognized that afrequent result of receptor-mediated responses to extracellular signalswas the transcriptional activation or inactivation of specific genesafter exposure of the cognate receptor to an extracellular signal thatinduces such activity. Thus, transcription of genes controlled byreceptor-responsive transcriptional elements often reflects the activityof the surface protein by virtue of transduction of an intracellularsignal.

To illustrate, the intracellular signal that is transduced can beinitiated by the specific interaction of an extracellular signal,particularly a ligand, with a cell surface receptor on the cell. Thisinteraction sets in motion a cascade of intracellular events, theultimate consequence of which is a rapid and detectable change in thetranscription or translation of a gene. By selecting transcriptionalregulatory sequences that are responsive to the transduced intracellularsignals and operatively linking the selected promoters to reportergenes, whose transcription, translation or ultimate activity is readilydetectable and measurable, the transcription based assay provides arapid indication of whether a specific receptor or ion channel interactswith a test peptide in any way that influences intracellulartransduction. Expression of the reporter gene, thus, provides a valuablescreening tool for the development of compounds that act as agonists orantagonists of a cell receptor or ion channel.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on receptor signaling. Typically, thereporter gene construct will include a reporter gene in operativelinkage with one or more transcriptional regulatory elements responsiveto the signal transduction activity of the target receptor, with thelevel of expression of the reporter gene providing thereceptor-dependent detection signal. The amount of transcription fromthe reporter gene may be measured using any method known to those ofskill in the art to be suitable. For example, specific mRNA expressionmay be detected using Northern blots or specific protein product may beidentified by a characteristic stain or an intrinsic activity.

In preferred embodiments, the gene product of the reporter is detectedby an intrinsic activity associated with that product. For instance, thereporter gene may encode a gene product that, by enzymatic activity,gives rise to a detection signal based on color, fluorescence, orluminescence.

The amount of expression from the reporter gene is then compared to theamount of expression in either the same cell in the absence of the testcompound or it may be compared with the amount of transcription in asubstantially identical cell that lacks the specific receptors. Acontrol cell may be derived from the same cells from which therecombinant cell was prepared but which had not been modified byintroduction of heterologous DNA, e.g., the encoding the testpolypeptide. Alternatively, it may be a cell in which the specificreceptors are removed. Any statistically or otherwise significantdifference in the amount of transcription indicates that the testpolypeptide has in some manner altered the activity of the specificreceptor.

In other preferred embodiments, the reporter or marker gene provides aselection method such that cells in which the peptide is a ligand forthe receptor have a growth advantage. For example the reporter couldenhance cell viability, relieve a cell nutritional requirement, and/orprovide resistance to a drug.

With respect to the target receptor, it may be endogenously expressed bythe host cell, or it may be expressed from a heterologous gene that hasbeen introduced into the cell. Methods for introducing heterologous DNAinto eukaryotic cells are of course well known in the art and any suchmethod may be used. In addition, DNA encoding various receptor proteinsis known to those of skill in the art or it may be cloned by any methodknown to those of skill in the art. In certain embodiments, such as whenan exogenous receptor is expressed, it may be desirable to inactivate,such as by deletion, a homologous receptor present in the cell.

The subject assay is useful for identifying polypeptides that interactwith any receptor protein whose activity ultimately induces a signaltransduction cascade in the host cell which can be exploited to producea detectable signal. In particular, the assays can be used to testfunctional ligand-receptor or ligand-ion channel interactions for cellsurface-localized receptors and channels, and also for cytoplasmic andnuclear receptors. As described in more detail below, the subject assaycan be used to identify effectors of, for example, G protein-coupledreceptors, receptor tyrosine kinases, cytokine receptors, and ionchannels, as well as steroid hormone receptors. In preferred embodimentsthe method described herein is used for identifying ligands for “orphanreceptors” for which no ligand is known.

In embodiments in which cell surface receptors are the assay targets, itwill be desirable for each of the peptides of the peptide library toinclude a signal sequence for secretion, e.g., which will ensureappropriate transport of the peptide to the endoplasmic reticulum, thegolgi, and ultimately to the cell surface so that it is able to interactwith cell surface receptors. In the case of yeast cells, the signalsequence will transport peptides to the periplasmic space.

Any transfectable cell that can express the desired cell surface proteinin a manner such the protein functions to intracellularly transduce anextracellular signal may be used. The cells may be selected such thatthey endogenously express the target receptor protein or may begenetically engineered to do so.

The preparation of cells which express the orphan FPRL1 receptor, apeptide library, and a reporter gene expression construct, aredescribed. These cells have been used to identify a novel ligand forthis receptor. The cells for the identification of receptor ligands andin drug screening assays to discover agents capable of modulatingreceptor activity.

Any cell surface protein that is known to those of skill in the art orthat may be identified by those of skill in the art may used in theassay. The cell surface protein may endogenously expressed on theselected cell or it may be expressed from cloned DNA.

II. Host Cells

Suitable host cells for generating the subject assay includeprokaryotes, yeast, or higher eukaryotic cells, especially mammaliancells. Prokaryotes include gram negative or gram positive organisms.Examples of suitable mammalian host cell lines include the COS-7 line ofmonkey kidney cells (ATCC CRL 1651) (Gluzman (1981) Cell 23:175) CV-1cells (ATCC CCL 70), L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines.

If yeast cells are used, the yeast may be of any species which arecultivable and in which an exogenous receptor can be made to engage theappropriate signal transduction machinery of the host cell. Suitablespecies include Kluyverei lactis, Schizosaccharomyces pombe, andUstilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeastwhich can be used in practicing the present invention are Neurosporacrassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris,Candida tropicalis, and Hansenula polymorpha. The term “yeast”, as usedherein, includes not only yeast in a strictly taxonomic sense, i.e.,unicellular organisms, but also yeast-like multicellular fungi orfilamentous fungi.

The choice of appropriate host cell will also be influenced by thechoice of detection signal. For instance, reporter constructs, asdescribed below, can provide a selectable or screenable trait upontranscriptional activation (or inactivation) in response to a signaltransduction pathway coupled to the target receptor. The reporter genemay be an unmodified gene already in the host cell pathway, such as thegenes responsible for growth arrest in yeast. It may be a host cell genethat has been operably linked to a “receptor-responsive” promoter.Alternatively, it may be a heterologous gene that has been so linked.Suitable genes and promoters are discussed below. In other embodiments,second messenger generation can be measured directly in the detectionstep, such as mobilization of intracellular calcium or phospholipidmetabolism are quantitated. Accordingly, it will be understood that toachieve selection or screening, the host cell must have an appropriatephenotype. For example, introducing a pheromone-responsive chimeric HIS3gene into a yeast that has a wild-type HIS3 gene would frustrate geneticselection. Thus, to achieve nutritional selection, an auxotrophic strainis wanted.

To further illustrate, in a preferred embodiment of the subject assayusing a yeast host cell, the yeast cells possess one or more of thefollowing characteristics: (a) the endogenous FUS1 gene has beeninactivated; (b) the endogenous SST2 gene, and/or other genes involve indesensitization, has been inactivated; (c) if there is a homologous,endogenous receptor gene it has been inactivated; and (d) if the yeastproduces an endogenous ligand to the exogenous receptor, the genesencoding for the ligand been inactivated.

Other complementations for use in the subject assay can be constructedwithout any undue experimentation. Indeed, many yeast geneticcomplementation with mammalian signal transduction proteins have beendescribed in the art. For example, Mosteller et al. (1994) Mol Cell Biol14:1104–12 demonstrates that human Ras proteins can complement loss ofras mutations in S. cerevisiae. Moreover, Toda et al. (1986) PrincessTakamatsu Symp 17: 253–60 have shown that human ras proteins cancomplement the loss of RAS1 and RAS2 proteins in yeast, and hence arefunctionally homologous. Both human and yeast RAS proteins can stimulatethe magnesium and guanine nucleotide-dependent adenylate cyclaseactivity present in yeast membranes. Ballester et al. (1989) Cell 59:681–6 describe a vector to express the mammalian GAP protein in theyeast S. cerevisiae. When expressed in yeast, GAP inhibits the functionof the human ras protein, and complements the loss of IRA1. IRA1 is ayeast gene that encodes a protein with homology to GAP and acts upstreamof RAS. Mammalian GAP can therefore function in yeast and interact withyeast RAS. Wei et al. (1994) Gene 151: 279–84 describes that a humanRas-specific guanine nucleotide-exchange factor, Cdc25GEF, cancomplement the loss of CDC25 function in S. cerevisiae. Martegani et al.(1992) EMBO J. 11: 2151–7 describe the cloning by functionalcomplementation of a mouse cDNA encoding a homolog of CDC25, aSaccharomyces cerevisiae RAS activator. Vojtek et al. (1993) J Cell Sci105: 777–85 and Matviw et al. (1992) Mol Cell Biol 12: 5033–40 describehow a mouse CAP protein, e.g., an adenylyl cyclase associated proteinassociated with ras-mediated signal transduction, can complementsdefects in S. cerevisiae. Papasavvas et al. (1992) Biochem Biophys ResCommun 184:1378–85 also suggest that inactivated yeast adenyl cyclasecan be complemented by a mammalian adenyl cyclase gene. Hughes et al.(1993) Nature 364: 349–52 describe the complementation of byr1 infission yeast by mammalian MAP kinase kinase (MEK). Parissenti et al.(1993) Mol Cell Endocrinol 98: 9–16 describes the reconstitution ofbovine protein kinase C (PKC) in yeast. The Ca(2+)- andphospholipid-dependent Ser/Thr kinase PKC plays important roles in thetransduction of cellular signals in mammalian cells. Marcus et al.(1995) PNAS 92: 6180–4 suggests the complementation of shk1 nullmutations in S. pombe by the either the structurally related S.cerevisiae Ste20 or mammalian p65PAK protein kinases.

“Inactivation”, with respect to genes of the host cell, means thatproduction of a functional gene product is prevented or inhibited.Inactivation may be achieved by deletion of the gene, mutation of thepromoter so that expression does not occur, or mutation of the codingsequence so that the gene product is inactive. Inactivation may bepartial or total.

“Complementation”, with respect to genes of the host cell, means that atleast partial function of inactivated gene of the host cell is suppliedby an exogenous nucleic acid. For instance, yeast cells can be“mammalianized”, and even “humanized”, by complementation of receptorand signal transduction proteins with mammalian homologs. To illustrate,inactivation of a yeast Byr2/Ste11 gene can be complemented byexpression of a human MEKK gene.

III. Expression Systems

Ligating a polynucleotide coding sequence into a gene construct, such asan expression vector, and transforming or transfecting into hosts,either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic(bacterial cells), are standard procedures used in producing otherwell-known proteins, including sequences encoding exogenous receptor andpeptide libraries. Similar procedures, or modifications thereof, can beemployed to prepare recombinant reagent cells of the present inventionby tissue-culture technology in accord with the subject invention.

In general, it will be desirable that the vector be capable ofreplication in the host cell. It may be a DNA which is integrated intothe host genome, and thereafter is replicated as a part of thechromosomal DNA, or it may be DNA which replicates autonomously, as inthe case of a plasmid. In the latter case, the vector will include anorigin of replication which is functional in the host. In the case of anintegrating vector, the vector may include sequences which facilitateintegration, e.g., sequences homologous to host sequences, or encodingintegrases.

Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are known in the art, andare described in, for example, Powels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985). Mammalian expression vectorsmay comprise non-transcribed elements such as an origin of replication,a suitable promoter and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking nontranscribed sequences, and 5′ or 3′nontranslated sequences, such as necessary ribosome binding sites, apoly-adenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

The transcriptional and translational control sequences in expressionvectors to be used in transforming mammalian cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication (Fiers et al.(1978) Nature 273:111) Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the Bgl I site locatedin the viral origin of replication is included. Exemplary vectors can beconstructed as disclosed by Okayama and Berg (1983, Mol. Cell Biol.3:280). A useful system for stable high level expression of mammalianreceptor cDNAs in C127 murine mammary epithelial cells can beconstructed substantially as described by Cosman et al (1986, Mol.Immunol. 23:935). Other expression vectors for use in mammalian hostcells are derived from retroviruses.

In other embodiments, the use of viral transfection can provide stablyintegrated copies of the expression construct. In particular, the use ofretroviral, adenoviral or adeno-associated viral vectors is contemplatedas a means for providing a stably transfected cell line which expressesan exogenous receptor, and/or a polypeptide library.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. Moreover, if yeast are used as a host cell, it will be understoodthat the expression of a gene in a yeast cell requires a promoter whichis functional in yeast. Suitable promoters include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Req. 7, 149 (1968); and Holland et al. Biochemistry 17, 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phospho-fructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedmetallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well asenzymes responsible for maltose and galactose utilization. Finally,promoters that are active in only one of the two haploid mating typesmay be appropriate in certain circumstances. Among thesehaploid-specific promoters, the pheromone promoters MFal and MFα1 are ofparticular interest.

In some instances, it may be desirable to derive the host cell usinginsect cells. In such embodiments, recombinant polypeptides can beexpressed by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

Libraries of random peptides or cDNA fragments may be expressed in amultiplicity of ways, including as portions of chimeric proteins. Asdescribed below, where secretion of the peptide library is desired, thepeptide library can be engineered for secretion or transport to theextracellular space via the yeast pheromone system.

In constructing suitable expression plasmids, the termination sequencesassociated with these genes, or with other genes which are efficientlyexpressed in yeast, may also be ligated into the expression vector 3′ ofthe heterologous coding sequences to provide polyadenylation andtermination of the mRNA.

IV. Periplasmic Secretion

If yeast cells are used as the host cell it will be noted that the yeastcell is bounded by a lipid bilayer called the plasma membrane. Betweenthis plasma membrane and the cell wall is the periplasmic space.Peptides secreted by yeast cells cross the plasma membrane through avariety of mechanisms and thereby enter the periplasmic space. Thesecreted peptides are then free to interact with other molecules thatare present in the periplasm or displayed on the outer surface of theplasma membrane. The peptides then either undergo re-uptake into thecell, diffuse through the cell wall into the medium, or become degradedwithin the periplasmic space.

The test polypeptide library may be secreted into the periplasm by anyof a number of exemplary mechanisms, depending on the nature of theexpression system to which they are linked. In one embodiment, thepeptide may be structurally linked to a yeast signal sequence, such asthat present in the α-factor precursor, which directs secretion throughthe endoplasmic reticulum and Golgi apparatus. Since this is the sameroute that the receptor protein follows in its journey to the plasmamembrane, opportunity exists in cells expressing both the receptor andthe peptide library for a specific peptide to interact with the receptorduring transit through the secretory pathway. This has been postulatedto occur in mammalian cells exhibiting autocrine activation. Suchinteraction could yield activation of the response pathway duringtransit, which would still allow identification of those cellsexpressing a peptide agonist. For situations in which peptideantagonists to externally applied receptor agonist are sought, thissystem would still be effective, since both the peptide antagonist andreceptor would be delivered to the outside of the cell in concert. Thus,those cells producing an antagonist would be selectable, since thepeptide antagonist would be properly and timely situated to prevent thereceptor from being stimulated by the externally applied agonist.

An alternative mechanism for delivering peptides to the periplasmicspace is to use the ATP-dependent transporters of the STE6/MDR1 class.This transport pathway and the signals that direct a protein or peptideto this pathway are not as well characterized as is the endoplasmicreticulum-based secretory pathway. Nonetheless, these transportersapparently can efficiently export certain peptides directly across theplasma membrane, without the peptides having to transit the ER/Golgipathway. It is anticipated that at least a subset of peptides can besecreted through this pathway by expressing the library in context ofthe a-factor prosequence and terminal tetrapeptide. The possibleadvantage of this system is that the receptor and peptide do not comeinto contact until both are delivered to the external surface of thecell. Thus, this system strictly mimics the situation of an agonist orantagonist that is normally delivered from outside the cell. Use ofeither of the described pathways is within the scope of the invention.

The present invention does not require periplasmic secretion, or, ifsuch secretion is provided, any particular secretion signal or transportpathway.

V. Cytokine Receptors

In one embodiment the target receptor is a cytokine receptor. Cytokinesare a family of soluble mediators of cell-to-cell communication thatincludes interleukins, interferons, and colony-stimulating factors. Thecharacteristic features of cytokines lie in their functional redundancyand pleiotropy. Most of the cytokine receptors that constitute distinctsuperfamilies do not possess intrinsic protein tyrosine kinase domains,yet receptor stimulation usually invokes rapid tyrosine phosphorylationof intracellular proteins, including the receptors themselves. Manymembers of the cytokine receptor superfamily acitvate the Jak proteintyrosine kinase family, with resultant phosphorylation of the STATtranscriptional activator factors. IL-2, IL-7, IL-2 and Interferon γhave all been shown to activate Jak kinases (Frank et al (1995) ProcNatl Acad Sci USA 92:7779–7783); Scharfe et al. (1995) Blood86:2077–2085); (Bacon et al. (1995) Proc Natl Acad Sci USA92:7307–7311); and (Sakatsume et al (1995) J. Biol Chem270:17528–17534). Events downstream of Jak phosphorylation have alsobeen elucidated. For example, exposure of T lymphocytes to IL-2 has beenshown to lead to the phosphorylation of signal transducers andactivators of transcription (STAT) proteins STAT1α, STAT2β, and STAT3,as well as of two STAT-related proteins, p94 and p95. The STAT proteinswere found to translocate to the nucleus and to bind to a specific DNAsequence, thus suggesting a mechanism by which IL-2 may activatespecific genes involved in immune cell function (Frank et al. supra).Jak3 is associated with the gamma chain of the IL-2, IL-4, and IL-7cytokine receptors (Fujii et al. (1995) Proc Natl Acad Sci 92:5482–5486)and (Musso et al (1995) J Exp Med. 181:1425–1431). The Jak kinases havealso been shown to be activated by numerous ligands that signal viacytokine receptors such as, growth hormone and erythropoietin and IL-6(Kishimoto (1994) Stem cells Suppl 12:37–44).

Detection signals which may be scored for in the present assay, inaddition to direct detection of second messangers, such as by changes inphosphorylation, includes reporter constructs which includetranscriptional regulatory elements responsive to the STAT proteins.Described infra.

VI. Multisubunit Immune Recognition Receptor (MIRR)

In another embodiment the receptor is a multisubunit receptor. Receptorscan be comprised of multiple proteins referred to as subunits, onecategory of which is referred to as a multisubunit receptor is amultisubunit immune recognition receptor (MIRR). MIRRs include receptorshaving multiple noncovalently associated subunits and are capable ofinteracting with src-family tyrosine kinases. MIRRs can include, but arenot limited to, B cell antigen receptors, T cell antigen receptors, Fcreceptors and CD22. One example of an MIRR is an antigen receptor on thesurface of a B cell. To further illustrate, the MIRR on the surface of aB cell comprises membrane-bound immunoglobulin (mIg) associated with thesubunits Ig-α and Ig-β or Ig-γ, which forms a complex capable ofregulating B cell function when bound by antigen. An antigen receptorcan be functionally linked to an amplifier molecule in a manner suchthat the amplifier molecule is capable of regulating gene transcription.

Src-family tyrosine kinases are enzymes capable of phosphorylatingtyrosine residues of a target molecule. Typically, a src-family tyrosinekinase contains one or more binding domains and a kinase domain. Abinding domain of a src-family tyrosine kinase is capable of binding toa target molecule and a kinase domain is capable of phosphorylating atarget molecule bound to the kinase. Members of the src family oftyrosine kinases are characterized by an N-terminal unique regionfollowed by three regions that contain different degrees of homologyamong all the members of the family. These three regions are referred toas src homology region 1 (SH1), src homology region 2 (SH2) and srchomology region 3 (SH3). Both the SH2 and SH3 domains are believed tohave protein association functions important for the formation of signaltransduction complexes. The amino acid sequence of an N-terminal uniqueregion, varies between each src-family tyrosine kinase. An N-terminalunique region can be at least about the first 40 amino acid residues ofthe N-terminal of a src-family tyrosine kinase.

Syk-family kinases are enzymes capable of phosphorylating tyrosineresidues of a target molecule. Typically, a syk-family kinase containsone or more binding domains and a kinase domain. A binding domain of asyk-family tyrosine kinase is capable of binding to a target moleculeand a kinase domain is capable of phosphorylating a target moleculebound to the kinase. Members of the syk- family of tyrosine kinases arecharacterized by two SH2 domains for protein association function and atyrosine kinase domain.

A primary target molecule is capable of further extending a signaltransduction pathway by modifying a second messenger molecule. Primarytarget molecules can include, but are not limited to,phosphatidylinositol 3-kinase (PI-3K), P21^(ras)GAPase-activatingprotein and associated P190 and P62 protein, phospholipases such asPLCγ1 and PLCγ2, MAP kinase, Shc and VAV. A primary target molecule iscapable of producing second messenger molecule which is capable offurther amplifying a transduced signal. Second messenger moleculesinclude, but are not limited to diacylglycerol and inositol1,4,5-triphosphate (IP3). Second messenger molecules are capable ofinitiating physiological events which can lead to alterations in genetranscription. For example, production of IP3 can result in release ofintracellular calcium, which can then lead to activation of calmodulinkinase II, which can then lead to serine phosphorylation of a DNAbinding protein referred to as ets-1 proto-onco-protein. Diacylglycerolis capable of activating the signal transduction protein, protein kinaseC which affects the activity of the AP1 DNA binding protein complex.Signal transduction pathways can lead to transcriptional activation ofgenes such as c-fos, egr-1, and c-myc.

Shc can be thought of as an adaptor molecule. An adaptor moleculecomprises a protein that enables two other proteins to form a complex(e.g., a three molecule complex). Shc protein enables a complex to formwhich includes Grb2 and SOS. Shc comprises an SH2 domain that is capableof associating with the SH2 domain of Grb2.

Molecules of a signal transduction pathway can associate with oneanother using recognition sequences. Recognition sequences enablespecific binding between two molecules. Recognition sequences can varydepending upon the structure of the molecules that are associating withone another. A molecule can have one or more recognition sequences, andas such can associate with one or more different molecules.

Signal transduction pathways for MIRR complexes are capable ofregulating the biological functions of a cell. Such functions caninclude, but are not limited to the ability of a cell to grow, todifferentiate and to secrete cellular products. MIRR-induced signaltransduction pathways can regulate the biological functions of specifictypes of cells involved in particular responses by an animal, such asimmune responses, inflammatory responses and allergic responses. Cellsinvolved in an immune response can include, for example, B cells, Tcells, macrophages, dendritic cells, natural killer cells and plasmacells. Cells involved in inflammatory responses can include, forexample, basophils, mast cells, eosinophils, neutrophils andmacrophages. Cells involved in allergic responses can include, forexample mast cells, basophils, B cells, T cells and macrophages.

In exemplary embodiments of the subject assay, the detection signal is asecond messangers, such as a phosphorylated src-like protein, includesreporter constructs which include transcriptional regulatory elementssuch as serum response element (SRE),12-O-tetradecanoyl-phorbol-13-acetate response element, cyclic AMPresponse element, c- fos promoter, or a CREB-responsive element.

VII. Nuclear Receptors

In another embodiment, the target receptor is a nuclear receptor. Thenuclear receptors may be viewed as ligand-dependent transcriptionfactors. These receptors provide a direct link between extracellularsignals, mainly hormones, and transcriptional responses. Theirtranscriptional activation fuction is regulated by endogenous smallmolecules, such as steroid hormones, vitamin D, ecdysone, retinoic acidsand thyroid hormones, which pass readily through the plasma membrane andbind their receptors inside the cell (Laudet and Adelmant (1995) CurrentBiology 5:124). The majority of these receptors appear to contain threedomains: a variable amino terminal domain; a highly conserved,DNA-binding domain and a moderately conserved, carboxyl-terminalligand-binding domain (Power et al. (1993) Curr. Opin. Cell Biol.5:499–504). Examples include the estrogen, progesterone, androgen,thyroid hormone and mineralocorticoid receptors. In addition to theknown steroid receptors, at least 40 orphan members of this superfamilyhave been identified. (Laudet et al., (1992) EMBO J. 11:1003–1013).There are at least four groups of orphan nuclear receptors representedby NGF1, FTZ-F1, Rev-erbs, and RARs, which are by evolutionarystandards, only distantly related to each other (Laudet et al. supra).While the steroid hormone receptors bind exclusively as homodimers to apalindrome of their hormone responsive element other nuclear receptorsbind as heterodimers. Interestingly, some orphan receptors bind asmonomers to similar response elements and require for their function aspecific motif that is rich in basic amino-acid residues and is locatedcorboxy-terminal to the DNA-binding domain (Laudet and Adelmant supra.)

In preferred embodiments, the subject assay is derived to utilize ahormone-dependent reporter construct for selection. For instance,glucocorticoid response elements (GREs) and thyroid receptorenhancer-like DNA sequences (TREs) can be used to drive expression ofreporter construct in response to hormone binding to hormone receptors.GRE's are enhancer-like DNA sequences that confer glucocorticoidresponsiveness via interaction with the glucocorticoid receptor. SeePayvar, et al. (1983) Cell 35:381 and Schiedereit et al. (1983) Nature304:749. TRE's are similar to GRE's except that they confer thyroidhormone responsiveness via interaction with thyroid hormone receptor.Turning now to the interaction of hormones and receptors, it is knownthat a steroid or thyroid hormone enters cells by facilitated diffusionand binds to its specific receptor protein, initiating an allostericalteration of the protein. As a result of this alteration, thehormone/receptor complex is capable of binding to certain specific siteson transcriptional regulatory sequence with high affinity.

It is also known that many of the primary effects of steroid and thyroidhormones involve increased transcription of a subset of genes inspecific cell types. Moreover, there is evidence that activation oftranscription (and, consequently, increased expression) of genes whichare responsive to steroid and thyroid hormones (through interaction ofchromatin with hormone receptor/hormone complex) is effected throughbinding of the complex to enhancers associated with the genes.

In any case, a number of steroid hormone and thyroid hormone responsivetranscriptional control units, some of which have been shown to includeenhancers, have been identified. These include the mouse mammary tumorvirus 5′-long terminal repeat (MMTV LTR), responsive to glucocorticoid,aldosterone and androgen hormones; the transcriptional control units formammalian growth hormone genes, responsive to glucocorticoids,estrogens, and thyroid hormones; the transcriptional control units formammalian prolactin genes and progesterone receptor genes, responsive toestrogens; the transcriptional control units for avian ovalbumin genes,responsive to progesterones; mammalian metallothionein genetranscriptional control units, responsive to glucocorticoids; andmammalian hepatic alpha 2u-globulin gene transcriptional control units,responsive to androgens, estrogens, thyroid hormones andglucocorticoids. Such steroid hormone and thyroid hormone responsivetranscriptional control units can be used to generate reporterconstructs which are sensitive to agonists and antagonists of thesteroid hormone and/or thyroid hormone receptors. See, for example, U.S.Pat. Nos. 5,298,429 and 5,071,773, both to Evans, et. al. Moreover, theart describes the functional expression of such receptors in yeast. Seealso for example, Caplan et al. (1995) J Biol Chem 270:5251–7; andBaniahmad et al. (1995) Mol Endocrinol 9: 34–43.

VIII. Receptor Tyrosine Kinases

In still another embodiment, the target receptor is a receptor tyrosinekinase. The receptor tyrosine kinases can be divided into five subgroupson the basis of structural similarities in their extracellular domainsand the organization of the tyrosine kinase catalytic region in theircytoplasmic domains. Sub-groups I (epidermal growth factor (EGF)receptor-like), II (insulin receptor-like) and the eph/eck familycontain cysteine-rich sequences (Hirai et al., (1987) Science238:1717–1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol.10:6316–6324). The functional domains of the kinase region of thesethree classes of receptor tyrosine kinases are encoded as a contiguoussequence (Hanks et al. (1988) Science 241:42–52). Subgroups III(platelet-derived growth factor (PDGF) receptor-like) and IV (thefibro-blast growth factor (FGF) receptors) are characterized as havingimmunoglobulin (Ig)-like folds in their extracellular domains, as wellas having their kinase domains divided in two parts by a variablestretch of unrelated amino acids (Yanden and Ullrich (1988) supra andHanks et al. (1988) supra).

The family with by far the largest number of known members is the EPHfamily. Since the description of the prototype, the EPH receptor (Hiraiet al. (1987) Science 238:1717–1720), sequences have been reported forat least ten members of this family, not counting apparently orthologousreceptors found in more than one species. Additional partial sequences,and the rate at which new members are still being reported, suggest thefamily is even larger (Maisonpierre et al. (1993) Oncogene 8:3277–3288;Andres et al. (1994) Oncogene 9:1461–1467; Henkemeyer et al. (1994)Oncogene 9:1001–1014; Ruiz et al. (1994) Mech Dev 46:87–100; Xu et al.(1994) Development 120:287–299; Zhou et al. (1994) J Neurosci Res37:129–143; and references in Tuzi and Gullick (1994) Br J Cancer69:417–421). Remarkably, despite the large number of members in the EPHfamily, all of these molecules were identified as orphan receptorswithout known ligands.

The expression patterns determined for some of the EPH family receptorshave implied important roles for these molecules in early vertebratedevelopment. In particular, the timing and pattern of expression of sek,mek4 and some of the other receptors during the phase of gastrulationand early organogenesis has suggested functions for these receptors inthe important cellular interactions involved in patterning the embryo atthis stage (Gilardi-Hebenstreit et al. (1992) Oncogene 7:2499–2506;Nieto et al. (1992) Development 116:1137–1150; Henkemeyer et al., supra;Ruiz et al., supra; and Xu et al., supra). Sek, for example, shows anotable early expression in the two areas of the mouse embryo that showobvious segmentation, namely the somites in the mesoderm and therhombomeres of the hindbrain; hence the name sek, for segmentallyexpressed kinase (Gilardi-Hebenstreit et al., supra; Nieto et al.,supra). As in Drosophila, these segmental structures of the mammalianembryo are implicated as important elements in establishing the bodyplan. The observation that Sek expression precedes the appearance ofmorphological segmentation suggests a role for sek in forming thesesegmental structures, or in determining segment-specific cell propertiessuch as lineage compartmentation (Nieto et al., supra). Moreover, EPHreceptors have been implicated, by their pattern of expression, in thedevelopment and maintenance of nearly every tissue in the embryonic andadult body. For instance, EPH receptors have been detected throughoutthe nervous system, the testes, the cartilaginous model of the skeleton,tooth primordia, the infundibular component of the pituitary, variousepithelia tissues, lung, pancreas, liver and kidney tissues.Observations such as this have been indicative of important and uniqueroles for EPH family kinases in development and physiology, but furtherprogress in understanding their action has been severely limited by thelack of information on their ligands.

As used herein, the terms “EPH receptor” or “EPH-type receptor” refer toa class of receptor tyrosine kinases, comprising at least elevenparalogous genes, though many more orthologs exist within this class,e.g. homologs from different species. EPH receptors, in general, are adiscrete group of receptors related by homology and easily reconizable,e.g., they are typically characterized by an extracellular domaincontaining a characteristic spacing of cysteine residues near theN-terminus and two fibronectin type III repeats (Hirai et al. (1987)Science 238:1717–1720; Lindberg et al. (1990) Mol Cell Biol10:6316–6324; Chan et al. (1991) Oncogene 6:1057–1061; Maisonpierre etal. (1993) Oncogene 8:3277–3288; Andres et al. (1994) Oncogene9:1461–1467; Henkemeyer et al. (1994) Oncogene 9:1001–1014; Ruiz et al.(1994) Mech Dev 46:87–100; Xu et al. (1994) Development 120:287–299;Zhou et al. (1994) J Neurosci Res 37:129–143; and references in Tuzi andGullick (1994) Br J Cancer 69:417–421). Exemplary EPH receptors includethe eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyrol, tyro4, tyro5,tyro6, tyro11, cek4, cek5, cek6, cek7, cek8, cek9, cek10, bsk, rtk1,rtk2, rtk3, myk1, myk2, ehk1, ehk2, pagliaccio, htk, erk and nukreceptors. The term “EPH receptor” refers to the membrane form of thereceptor protein, as well as soluble extracellular fragments whichretain the ability to bind the ligand of the present invention.

In exemplary embodiments, the detection signal is provided by detectingphosphorylation of intracellular proteins, e.g., MEKKs, MEKs, or Mapkinases, or by the use of reporter constructs which includetranscriptional regulatory elements responsive to c-fos and/or c-jun.Described infra.

IX. G Protein-Coupled Receptors

One family of signal transduction cascades found in eukaryotic cellsutilizes heterotrimeric “G proteins.” Many different G proteins areknown to interact with receptors. G protein signaling systems includethree components: the receptor itself, a GTP-binding protein (Gprotein), and an intracellular target protein.

The cell membrane acts as a switchboard. Messages arriving throughdifferent receptors can produce a single effect if the receptors act onthe same type of G protein. On the other hand, signals activating asingle receptor can produce more than one effect if the receptor acts ondifferent kinds of G proteins, or if the G proteins can act on differenteffectors.

In their resting state, the G proteins, which consist of alpha (α), beta(β) and gamma (γ) subunits, are complexed with the nucleotide guanosinediphosphate (GDP) and are in contact with receptors. When a hormone orother first messenger binds to receptor, the receptor changesconformation and this alters its interaction with the G protein. Thisspurs the α subunit to release GDP, and the more abundant nucleotideguanosine triphosphate (GTP), replaces it, activating the G protein. TheG protein then dissociates to separate the α subunit from the stillcomplexed beta and gamma subunits. Either the Gα subunit, or the Gβγcomplex, depending on the pathway, interacts with an effector. Theeffector (which is often an enzyme) in turn converts an inactiveprecursor molecule into an active “second messenger,” which may diffusethrough the cytoplasm, triggering a metabolic cascade. After a fewseconds, the Gα converts the GTP to GDP, thereby inactivating itself.The inactivated Gα may then reassociate with the Gβγ complex.

Hundreds, if not thousands, of receptors convey messages throughheterotrimeric G proteins, of which at least 17 distinct forms have beenisolated. Although the greatest variability has been seen in the αsubunit, several different β and γ structures have been reported. Thereare, additionally, several different G protein-dependent effectors.

Most G protein-coupled receptors are comprised of a single protein chainthat is threaded through the plasma membrane seven times. Such receptorsare often referred to as seven-transmembrane receptors (STRs). More thana hundred different STRs have been found, including many distinctreceptors that bind the same ligand, and there are likely many more STRsawaiting discovery.

In addition, STRs have been identified for which the natural ligands areunknown; these receptors are termed “orphan” G protein-coupledreceptors, as described above. Examples include receptors cloned byNeote et al. (1993) Cell 72, 415; Kouba et al. FEBS Lett. (1993) 321,173; Birkenbach et al.(1993) J. Virol. 67, 2209.

The “exogenous receptors” of the present invention may be any Gprotein-coupled receptor which is exogenous to the cell which is to begenetically engineered for the purpose of the present invention. Thisreceptor may be a plant or animal cell receptor. Screening for bindingto plant cell receptors may be useful in the development of, e.g.,herbicides. In the case of an animal receptor, it may be of invertebrateor vertebrate origin. If an invertebrate receptor, an insect receptor ispreferred, and would facilitate development of insecticides. Thereceptor may also be a vertebrate, more preferably a mammalian, stillmore preferably a human, receptor. The exogenous receptor is alsopreferably a seven transmembrane segment receptor.

Known ligands for G protein coupled receptors include: purines andnucleotides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin and thelike; biogenic amines (and related natural ligands), such as5-hydroxytryptamine, acetylcholine, dopamine, adrenaline, adrenaline,adrenaline, histamine, noradrenaline, noradrenaline, noradrenaline,tyramine/octopamine and other related compounds; peptides such asadrenocorticotrophic hormone (acth), melanocyte stimulating hormone(msh), melanocortins, neurotensin (nt), bombesin and related peptides,endothelins, cholecystokinin, gastrin, neurokinin b (nk3), invertebratetachykinin-like peptides, substance k (nk2), substance p (nk1),neuropeptide y (npy), thyrotropin releasing-factor (trf), bradykinin,angiotensin ii, beta-endorphin, c5a anaphalatoxin, calcitonin,chemokines (also called intercrines), corticotrophic releasing factor(crf), dynorphin, endorphin, fmlp and other formylated peptides,follitropin (fsh), fungal mating pheremones, galanin, gastric inhibitorypolypeptide receptor (gip), glucagon-like peptides (glps), glucagon,gonadotropin releasing hormone (gnrh), growth hormone releasinghormone(ghrh), insect diuretic hormone, interleukin-8, leutropin(lh/hcg), met-enkephalin, opioid peptides, oxytocin, parathyroid hormone(pth) and pthrp, pituitary adenylyl cyclase activiating peptide (pacap),secretin, somatostatin, thrombin, thyrotropin (tsh), vasoactiveintestinal peptide (vip), vasopressin, vasotocin; eicosanoids such asip-prostacyclin, pg-prostaglandins, α-thromboxanes; retinal basedcompounds such as vertebrate 11-cis retinal, invertebrate 11-cis retinaland other related compounds; lipids and lipid-based compounds such ascannabinoids, anandamide, lysophosphatidic acid, platelet activatingfactor, leukotrienes and the like; excitatory amino acids and ions suchas calcium ions and glutamate.

Suitable examples of G-protein coupled receptors include, but are notlimited to, dopaminergic, muscarinic cholinergic, a-adrenergic,b-adrenergic, opioid (including delta and mu), cannabinoid,serotoninergic, and GABAergic receptors. Preferred receptors include the5HT family of receptors, dopamine receptors, C5a receptor and FPRL-1receptor, cyclo-histidyl-proline-diketoplperazine receptors, melanocytestimulating hormone release inhibiting factor receptor, and receptorsfor neurotensin, thyrotropin releasing hormone, calcitonin,cholecytokinin-A, neurokinin-2, histamine-3, cannabinoid, melanocortin,or adrenomodulin, neuropeptide-Y1 or galanin. Other suitable receptorsare listed in the art. The term “receptor,” as used herein, encompassesboth naturally occurring and mutant receptors.

Many of these G protein-coupled receptors, like the yeast a- andα-factor receptors, contain seven hydrophobic amino acid-rich regionswhich are assumed to lie within the plasma membrane. Specific human Gprotein-coupled STRs for which genes have been isolated and for whichexpression vectors could be constructed include those listed herein andothers known in the art. Thus, the gene would be operably linked to apromoter functional in the cell to be engineered and to a signalsequence that also functions in the cell. For example in the case ofyeast, suitable promoters include Ste2, Ste3 and gal10. Suitable signalsequences include those of Ste2, Ste3 and of other genes which encodeproteins secreted by yeast cells. Preferably, when a yeast cell is used,the codons of the gene would be optimized for expression in yeast. SeeHoekema et al.,(1987) Mol. Cell. Biol., 7:2914–24; Sharp, et al.,(1986)14:5125–43.

The homology of STRs is discussed in Dohlman et al., Ann. Rev. Biochem.,(1991) 60:653–88. When STRs are compared, a distinct spatial pattern ofhomology is discernible. The transmembrane domains are often the mostsimilar, whereas the N- and C-terminal regions, and the cytoplasmic loopconnecting transmembrane segments V and VI are more divergent.

The functional significance of different STR regions has been studied byintroducing point mutations (both substitutions and deletions) and byconstructing chimeras of different but related STRs. Synthetic peptidescorresponding to individual segments have also been tested for activity.Affinity labeling has been used to identify ligand binding sites.

It is conceivable that a foreign receptor which is expressed in yeastwill functionally integrate into the yeast membrane, and there interactwith the endogenous yeast G protein. More likely, either the receptorwill need to be modified (e.g., by replacing its V–VI loop with that ofthe yeast STE2 or STE3 receptor), or a compatible G protein should beprovided.

If the wild-type exogenous G protein-coupled receptor cannot be madefunctional in yeast, it may be mutated for this purpose. A comparisonwould be made of the amino acid sequences of the exogenous receptor andof the yeast receptors, and regions of high and low homology identified.Trial mutations would then be made to distinguish regions involved inligand or G protein binding, from those necessary for functionalintegration in the membrane. The exogenous receptor would then bemutated in the latter region to more closely resemble the yeastreceptor, until functional integration was achieved. If this wereinsufficient to achieve functionality, mutations would next be made inthe regions involved in G protein binding. Mutations would be made inregions involved in ligand binding only as a last resort, and then aneffort would be made to preserve ligand binding by making conservativesubstitutions whenever possible.

Preferably, the yeast genome is modified so that it is unable to producethe yeast receptors which are homologous to the exogenous receptors infunctional form. Otherwise, a positive assay score might reflect theability of a peptide to activate the endogenous G protein-coupledreceptor, and not the receptor of interest.

A. Chemoattractant Receptors

The N-formyl peptide receptor is a classic example of a calciummobilizing G protein-coupled receptor expressed by neutrophils and otherphagocytic cells of the mammalian immune system (Snyderman et al. (1988)In Inflammation: Basic Principles and Clinical Correlates, pp. 309–323).N-formyl peptides of bacterial origin bind to the receptor and engage acomplex activation program that results in directed cell movement,release of inflammatory granule contents, and activation of a latentNADPH oxidase which is important for the production of metabolites ofmolecular oxygen. This pathway initiated by receptor-ligand interactionis critical in host protection from pyogenic infections. Similar signaltransduction occurs in response to the inflammatory peptides C5a andIL-8.

Two other formyl peptide receptor like (FPRL) genes have been clonedbased on their ability to hybridize to a fragment of the NFPR cDNAcoding sequence. These have been named FPRL1 (Murphy et al. (1992) J.Biol. Chem. 267:7637–7643) and FPRL2 (Ye et al. (1992) Biochem BiophysRes. Comm. 184:582–589). FPRL2 was found to mediate calcium mobilizationin mouse fibroblasts transfected with the gene and exposed to formylpeptide. In contrast, although FPRL1 was found to be 69% identical inamino acid sequence to NFPR, it did not bind prototype N-formyl peptidesligands when expressed in heterologous cell types. This lead to thehypothesis of the existence of an as yet unidentified ligand for theFPRL1 orphan receptor (Murphy et al. supra).

Using the technology described herein a ligand has been cloned for theseorphan receptors.

B. G Proteins

In the case of an exogenous G-protein coupled receptor, the yeast cellmust be able to produce a G protein which is activated by the exogenousreceptor, and which can in turn activate the yeast effector(s). The artsuggests that the endogenous yeast Gα subunit (e.g., GPA) will be oftenbe sufficiently homologous to the “cognate” Gα subunit which is nativelyassociated with the exogenous receptor for coupling to occur. Morelikely, it will be necessary to genetically engineer the yeast cell toproduce a foreign Gα subunit which can properly interact with theexogenous receptor. For example, the Gα subunit of the yeast G proteinmay be replaced by the Gα subunit natively associated with the exogenousreceptor.

Dietzel and Kurjan, (1987) Cell, 50:1001) demonstrated that rat Gαsfunctionally coupled to the yeast Gβγ complex. However, rat Gαi2complemented only when substantially overexpressed, while Gα0 did notcomplement at all. Kang, et al., Mol. Cell. Biol., (1990)10:2582).Consequently, with some foreign Gα subunits, it is not feasible tosimply replace the yeast Gα.

If the exogenous G protein coupled receptor is not adequately coupled toyeast Gβγ by the Gα subunit natively associated with the receptor, theGα subunit may be modified to improve coupling. These modificationsoften will take the form of mutations which increase the resemblance ofthe Gα subunit to the yeast Gα while decreasing its resemblance to thereceptor-associated Gα. For example, a residue may be changed so as tobecome identical to the corresponding yeast Gα residue, or to at leastbelong to the same exchange group of that residue. After modification,the modified Gα subunit might or might not be “substantially homologous”to the foreign and/or the yeast Gα subunit.

The modifications are preferably concentrated in regions of the Gα whichare likely to be involved in Gβγ binding. In some embodiments, themodifications will take the form of replacing one or more segments ofthe receptor-associated Gα with the corresponding yeast Gα segment(s),thereby forming a chimeric Gα subunit. (For the purpose of the appendedclaims, the term “segment” refers to three or more consecutive aminoacids.) In other embodiments, point mutations may be sufficient.

This chimeric Gα subunit will interact with the exogenous receptor andthe yeast Gβγ complex, thereby permitting signal transduction. While useof the endogenous yeast Gβγ is preferred, if a foreign or chimeric Gβγis capable of transducing the signal to the yeast effector, it may beused instead.

C. Gα Structure

Some aspects of Gα structure are relevant to the design of modified Gαsubunits. The amino terminal 66 residues of GPA1 are aligned with thecognate domains of human Gαs, Gαi2, Gαi3, Gα16 and transducin. In theGPA41Gα hybrids, the amino terminal 41 residues (derived from GPA1) areidentical, end with the sequence-LEKQRDKNE- (SEQ ID NO:99) and areunderlined for emphasis. All residues following the glutamate (E)residue at position 41 are contributed by the human Gα subunits,including the consensus nucleotide binding motif -GxGxxG-. Periods inthe sequences indicate gaps that have been introduced to maximizealignments in this region. Codon bias is mammalian. For alignments ofthe entire coding regions of GPA1 with Gαs, Gαi, and GαO, Gαq and Gαz,see Dietzel and Kurjan (1987, Cell 50:573) and Lambright, et al. (1994,Nature 369:621–628). Additional sequence information is provided byMattera, et al. (1986, FEBS Lett 206:36–41), Bray, et al. (1986, Proc.Natl. Acad. Sci USA 83:8893–8897) and Bray, et al. (1987, Proc Natl.Acad Sci USA 84:5115–5119).

The gene encoding a G protein homolog of S. cerevisiae was clonedindependently by Dietzel and Kurjan (supra) (SCGI) and by Nakafuku, etal. (1987 Proc Natl Acad Sci 84:2140–2144) (GPA1). Sequence analysisrevealed a high degree of homology between the protein encoded by thisgene and mammalian Gα. GPA1 encodes a protein of 472 amino acids, ascompared with approximately 340–350 a.a. for most mammalian Gα subunitsin four described families, Gαs, Gαi, Gαq and Gα12/13. Nevertheless,GPA1 shares overall sequence and structural homology with all Gαproteins identified to date. The highest overall homology in GPA1 is tothe Gαi family (48% identity, or 65% with conservative substitutions)and the lowest is to GQS (33% identity, or 51% withconservativesubstitutions) (Nakaftiku, et al., supra).

The regions of high sequence homology among Gα subunits are dispersedthroughout their primary sequences, with the regions sharing the highestdegree of homology mapping to sequence that comprises the guaninenucleotide binding/GTPase domain. This domain is structurally similar tothe aβ fold of ras proteins and the protein synthesis elongation factorEF-Tu. This highly conserved guanine nucleotide-binding domain consistsof a six-stranded β sheet surrounded by a set of five α-helices. It iswithin these β sheets and a helices that the highest degree ofconservation is observed among all Gα proteins, including GPA1. Theleast sequence and structural homology is found in the intervening loopsbetween the β sheets and α helices that define the core GTPase domain.There are a total of four “intervening loops” or “inserts” present inall Gα subunits. In the crystal structures reported to date for the GDP-and GTPγS-liganded forms of bovine rod transducin (Noel, et al. (1993)Nature 366:654–663); (Lambright, et al. (1994) Nature 369:621–628), theloop residues are found to be outside the core GTPase structure.Functional roles for these loop structures have been established in onlya few instances. A direct role in coupling to phosphodiesterase-γ hasbeen demonstrated for residues within inserts 3 and 4 of Gαt (Rarick, etal. (1992) Science 256:1031–1033); (Artemyev, et al. (1992) J. Biol.Chem. 267:25067–25072), while a “GAP-like” activity has been ascribed tothe largely α-helical insert 1 domain of Gαs (Markby, et al. (1993)Science 262:1805–1901).

While the amino- and carboxy-termini of Gα subunits do not sharestriking homology either at the primary, secondary, or tertiary levels,there are several generalizations that can be made about them. First,the amino termini of Gα subunits have been implicated in the associationof Gα with Gβγ complexes and in membrane association via N-terminalmyristoylation. In addition, the carboxy-termini have been implicated inthe association of Gαβγ heterotrimeric complexes with G protein-coupledreceptors (Sullivan, et al. (1987) Nature 330:758–760); West, et al.(1985) J. Biol. Chem. 260:14428–14430); (Conklin, et al. (1993) Nature363:274–276). Data in support of these generalizations about thefunction of the N-terminus derive from several sources, including bothbiochemical and genetic studies.

As indicated above, there is little if any sequence homology sharedamong the amino termini of Gα subunits. The amino terminal domains of Gαsubunits that precede the first β-sheet (containing the sequence motif-LLLLGAGESG- (SEQ ID NO:1); see Noel, et al. (supra) for the numberingof the structural elements of Gα subunits) vary in length from 41 aminoacids (GPA1) to 31 amino acids (Gat). Most Gα subunits share theconsensus sequence for the addition of myristic acid at their aminotermini (MGxxxS-), although not all Gα subunits that contain this motifhave myristic acid covalently associated with the glycine at position 2(Speigel, et al. (1991) TIBS 16:338–3441). The role of thispost-translational modification has been inferred from studies in whichthe activity of mutant Gα subunits from which the consensus sequence formyristoylation has been added or deleted has been assayed (Mumby et al.(1990) Proc. Natl. Acad. Sci. USA 87: 728–732; (Linder, et al. (1991) J.Biol. Chem. 266:4654–4659); Gallego, et al. (1992) Proc. Natl. Acad.Sci. USA 89:9695–9699). These studies suggest two roles for N-terminalmyristoylation. First, the presence of amino-terminal myristic acid hasin some cases been shown to be required for association of Gα subunitswith the membrane, and second, this modification has been demonstratedto play a role in modulating the association of Gα subunits with Gβγcomplexes. The role of myristoylation of the GPA1 gene products, atpresent, unknown.

In other biochemical studies aimed at examining the role of theamino-terminus of Gα in driving the association between Gα and Gβγsubunits, proteolytically or genetically truncated versions of Gαsubunits were assayed for their ability to associate with Gβγcomplexes,bind guanine nucleotides and/or to activate effector molecules. In allcases, Gα subunits with truncated amino termini were deficient in allthree functions (Graf, et al. (1992) J. Biol. Chem. 267:24307–24314);(Journot, et al. (1990) J. Biol. Chem. 265:9009–9015); and (Neer, et al.(1988) J. Biol. Chem 263:8996–9000). Slepak, et al. (1993, J. Biol.Chem. 268:1414–1423) reported a mutational analysis of the N-terminal 56a.a. of mammalian GαO expressed in Escherichia coli. Molecules with anapparent reduced ability to interact with exogenously added mammalianGβγ were identified in the mutant library. As the authors pointed out,however, the assay used to screen the mutants the extent ofADP-ribosylation of the mutant Gα by pertussis toxin was not acompletely satisfactory probe of interactions between Gα and Gβγ.Mutations identified as inhibiting the interaction of the subunits,using this assay, may still permit the complexing of Gα and Gβγ whilesterically hindering the ribosylation of Gα by toxin. Genetic studiesexamined the role of amino-terminal determinants of Gα in heterotrimersubunit association have been carried out in both yeast systems usingGPA1-mammalian Gα hybrids (Kang, et al. (1990) Mol. Cell Biol.10:2582–2590) and in mammalian systems using Gαi/Gαs hybrids (Russelland Johnson (1993) Mol. Pharmacol. 44:255–263). In the former studies,gene fusions, composed of yeast GPA1 and mammalian Gα sequences wereconstructed by Kang, et al. (supra) and assayed for their ability tocomplement a gpal null phenotype (i.e., constitutive activation of thepheromone response pathway) in S. cerevisiae. Kang, et al. demonstratedthat wild type mammalian Gαs, Gαi but not GαO proteins are competent toassociate with yeast Gα and suppress the gpal null phenotype, but onlywhen overexpressed. Fusion proteins containing the amino-terminal 330residues of GPA1 sequence linked to 160, 143, or 142 residues of themammalian Gαs, Gαi and Gαo carboxyl-terminal regions, respectively, alsocoupled to the yeast mating response pathway when overexpressed on highcopy plasmids with strong inducible (CUP) or constitutive (PGK)promoters. All three of these hybrid molecules were able to complementthe gpal null mutation in a growth arrest assay, and were additionallyable to inhibit αfactor responsiveness and mating in tester strains.These last two observations argue that hybrid yeast-mammalian Gαsubunits are capable of interacting directly with yeast Gβγ, therebydisrupting the normal function of the yeast heterotrimer. Fusionscontaining the amino terminal domain of Gαs, Gαi or GαO, however, didnot complement the gpal null phenotype, indicating a requirement fordeterminants in the amino terminal 330 amino acid residues of GPA1 forassociation and sequestration of yeast Gβγ complexes. Taken together,these data suggest that determinants in the amino terminal region of Gαsubunits determine not only the ability to associate with Gβγ subunitsin general, but also with specific Gβγ subunits in a species-restrictedmanner.

Hybrid Gαi/Gαs subunits have been assayed in mammalian expressionsystems (Russell and Johnson (supra). In these studies, a large numberof chimeric Gα subunits were assayed for an ability to activate adenylylcyclase, and therefore, indirectly, for an ability to interact withGβγ(i.e., coupling of Gα to Gβγ=inactive cyclase; uncoupling of Gα fromGβγ=active cyclase). From these studies a complex picture emerged inwhich determinants in the region between residues 25 and 96 of thehybrids were found to determine the state of activation of these allelesas reflected in their rates of guanine nucleotide exchange and GTPhydrolysis and the extent to which they activated adenylyl cyclase invivo. These data could be interpreted to support the hypothesis thatstructural elements in the region between the amino terminal methionineand the ˜1 sheet identified in the crystal structure of Gat (see Noel,et al. supra and Lambright, et al. supra) are involved in determiningthe state of activity of the heterotrimer by (1) drivingassociation/dissociation between Gαand Gβγ subunits; (2) driving GDP/GTPexchange. While there is no direct evidence provided by these studies tosupport the idea that residues in this region of Gα and residues in Gβγsubunits contact one another, the data nonetheless provide a positiveindication for the construction of hybrid Gα subunits that retainfunction. There is, however, a negative indicator that derives from thiswork in that some hybrid constructs resulted in constitutive activationof the chimeric proteins (i.e., a loss of receptor-dependent stimulationof Gβγ dissociation and effector activation).

D. Construction of Chimeric Gα Subunits

In designing Gα subunits capable of transmitting, in yeast, signalsoriginating at mammalian G protein-coupled receptors, two generaldesiderata were recognized. First, the subunits should retain as much ofthe sequence of the native mammalian proteins as possible. Second, thelevel of expression for the heterologous components should approach, asclosely as possible, the level of their endogenous counterparts. Theresults described by King, et al. (1990, Science 250:121–123) forexpression of the human β2-adrenergic receptor and Gαs in yeast, takentogether with negative results obtained by Kang, et al. (supra) withfull-length mammalian Gα subunits other than Gαs, led us to thefollowing preferences for the development of yeast strains in whichmammalian G protein-coupled receptors could be linked to the pheromoneresponse pathway.

1. Mammalian Gα subunits will be expressed using the native sequence ofeach subunit or, alternatively, as minimal gene fusions with sequencesfrom the amino- terminus of GPA1 replacing the homologous residues fromthe mammalian Gα subunits.

2. Mammalian Gα subunits will be expressed from the GPA1 promotor eitheron low copy plasmids or after integration into the yeast genome as asingle copy gene.

3. Endogenous Gβγ subunits will be provided by the yeast STE4 and STE18loci.

E. Site-Directed Mutagenesis Versus Random Mutagenesis

There are two general approaches to solving structure-function problemsof the sort presented by attempts to define the determinants involved inmediating the association of the subunits that comprise the G proteinheterotrimer. The first approach, discussed above with respect to hybridconstructs, is a rational one in which specific mutations or alterationsare introduced into a molecule based upon the available experimentalevidence. In a second approach, random mutagenesis techniques, coupledwith selection or screening systems, are used to introduce large numbersof mutations into a molecule, and that collection of randomly mutatedmolecules is then subjected to a selection for the desired phenotype ora screen in which the desired phenotype can be observed against abackground of undesirable phenotypes. With random mutagenesis one canmutagenize an entire molecule or one can proceed by cassettemutagenesis. In the former instance, the entire coding region of amolecule is mutagenized by one of several methods (chemical, PCR, dopedoligonucleotide synthesis) and that collection of randomly mutatedmolecules is subjected to selection or screening procedures. Randommutagenesis can be applied in this way in cases where the molecule beingstudied is relatively small and there are powerful and stringentselections or screens available to discriminate between the differentclasses of mutant phenotypes that will inevitably arise. In the secondapproach, discrete regions of a protein, corresponding either to definedstructural (i.e. α-helices, β-sheets, turns, surface loops) orfunctional determinants (e.g., catalytic clefts, binding determinants,transmembrane segments) are subjected to saturating or semi-randommutagenesis and these mutagenized cassettes are re-introduced into thecontext of the otherwise wild type allele. Cassette mutagenesis is mostuseful when there is experimental evidence available to suggest aparticular function for a region of a molecule and there is a powerfulselection and/or screening approach available to discriminate betweeninteresting and uninteresting mutants. Cassette mutagenesis is alsouseful when the parent molecule is comparatively large and the desire isto map the functional domains of a molecule by mutagenizing the moleculein a step-wise fashion, i.e. mutating one linear cassette of residues ata time and then assaying for function.

The present invention contemplates applying random mutagenesis in orderto further delineate the determinants involved in Gα-Gβγ association.Random mutagenesis may be accomplished by many means, including:

1. PCR mutagenesis, in which the error prone Taq polymerase is exploitedto generate mutant alleles of Gα subunits, which are assayed directly inyeast for an ability to couple to yeast Gβγ.

2. Chemical mutagenesis, in which expression cassettes encoding Gαsubunits are exposed to mutagens and the protein products of the mutantsequences are assayed directly in yeast for an ability to couple toyeast Gβγ.

3. Doped synthesis of oligonucleotides encoding portions of the Gα gene.

4. In vivo mutagenesis, in which random mutations are introduced intothe coding region of Gα subunits by passage through a mutator strain ofE. coli, XL1-Red (mutD5 mutS mutT) (Stratagene, Menasa, Wis.).

The random mutagenesis may be focused on regions suspected to beinvolved in Gα-Gβγ association as discussed in the next section. Randommutagenesis approaches are feasible for two reasons. First, in yeast onehas the ability to construct stringent screens and facile selections(growth vs. death, transcription vs. lack of transcription) that are notreadily available in mammalian systems. Second, when using yeast it ispossible to screen efficiently through thousands of transformantsrapidly. Cassette mutagenesis is immediately suggested by theobservation (see infra) that the GPA₄₁ hybrids couple to the pheromoneresponse pathway. This relatively small region of Gα subunits representsa reasonable target for this type of mutagenesis. Another region thatmay be amenable to cassette mutagenesis is that defining the surface ofthe switch region of Gα subunits that is solvent-exposed in the crystalstructures of Gαi and transducin. From the data described below, thissurface may contain residues that are in direct contact with yeast Gβγsubunits, and may therefore be a reasonable target for mutagenesis.

F. Rational Design of Chimeric Gα Subunits

Several classes of rationally designed GPA1-mammalian Gα hybrid subunitshave been tested for the ability to couple to yeast βγ. The first, andlargest, class of hybrids are those that encode different lengths of theGPA1 amino terminal domain in place of the homologous regions of themammalian Gα subunits. This class of hybrid molecules includesGPA_(BAMH1), GPA₄₁, GPA_(ID), and GPA_(LW) hybrids, described below. Therationale for constructing these hybrid Gα proteins is based on results,described above, that bear on the importance of the amino terminalresidues of Gα in mediating interaction with Gβγ.

Preferably, the yeast Gα subunit is replaced by a chimeric Gα subunit inwhich a portion, e.g., at least about 20, more preferably at least about40, amino acids, which is substantially homologous with thecorresponding residues of the amino terminus of the yeast Gα, is fusedto a sequence substantially homologous with the main body of a mammalian(or other exogenous) Gα. While 40 amino acids is the suggested startingpoint, shorter or longer portions may be tested to determine the minimumlength required for coupling to yeast Gβγ and the maximum lengthcompatible with retention of coupling to the exogenous receptor. It ispresently believed that only the final 10 or 20 amino acids at thecarboxy terminus of the Gα subunit are required for interaction with thereceptor.

GPA_(BAMH1) hybrids. Kang et al. supra. described hybrid G α subunitsencoding the amino terminal 310 residues of GPA1 fused to the carboxylterminal 160, 143 and 142 residues, respectively, of Gαs, Gαi2, and GαO.In all cases examined by Kang et al., the hybrid proteins were able tocomplement the growth arrest phenotype of gpal strains. We haveconfirmed these findings and, in addition, have constructed and testedhybrids between GPA1 and Gαi3, Gαq and Gα16. All hybrids of this typethat have been tested functionally complement the growth arrestphenotype of gpal strains.

GPA41 hybrids. The rationale for constructing a minimal hybrid encodingonly 41 amino acids of GPA1 relies upon the biochemical evidence for therole of the amino-terminus of Gα subunits discussed above, together withthe following observation. Gβ and Gγ subunits are known to interact viaα- helical domains at their respective amino-termini (Pronin, et al.(1992) Proc. Natl. Acad. Sci. USA 89:6220–6224); Garritsen, et al.1993). The suggestion that the amino termini of Gα subunits may form anhelical coil and that this helical coil may be involved in associationof Gα with Gβγ (Masters et al (1986) Protein Engineering 1:47–54); Lupaset al.(1992) FEBS Lett. 314:105–108) leads to the hypothesis that thethree subunits of the G-protein heterotrimer interact with one anotherreversibly through the winding and unwinding of their amino-terminalhelical regions. A mechanism of this type has been suggested, as well,from an analysis of leucine zipper mutants of the GCN4 transcriptionfactor (Harbury, et al. (1993) Science 262:1401–1407). The rationale forconstructing hybrids like those described by Kang, et al. supra., thatcontain a majority of yeast sequence and only minimal mammaliansequence, derives from their ability to function in assays of couplingbetween Gα and Gβγ subunits. However, these chimeras had never beenassayed for an ability to couple to both mammalian G protein-coupledreceptors and yeast Gβγ subunits, and hence to reconstitute a hybridsignaling pathway in yeast.

GPA₄₁ hybrids that have been constructed and tested include Gαs, Gαi2,Gαi3, Gαq, Gαo_(a), Gαo_(b) and Gα16. Hybrids of Gαs, Gαi2, Gαi3, andGα16 functionally complement the growth arrest phenotype of gpalstrains, while GPA₄₁ hybrids of Gαo_(a) and Gαo_(b) do not. In additionto being tested in a growth arrest assay, these constructs have beenassayed in the more sensitive transcriptional assay for activation of afuslp-HIS3 gene. In both of these assays, the GPA₄₁-Gαs hybrid couplesless well than the GPα₄₁-i2, -i3, and -16 hybrids, while theGPα₄₁-o_(a), and -o_(b) hyrids do not function in either assay.

Several predictive algorithms indicate that the amino terminal domain upto the highly conserved sequence motif-LLLLGAGESG- (SEQ ID NO:1) (thefirst L in this motif is residue 43 in GPA1) forms a helical structurewith amphipathic character. Assuming that a heptahelical repeat unit,the following hybrids between GPA1 and GαS can be used to define thenumber of helical repeats in this motif necessary for hybrid function:

GPAl-7/GαS8–394

GPAl-14/GαS1514 394

GPAl-2 l/GαS22–394

GPAl-28/GαS29–394

GPAl-35/GαS36–394

GPAl-42/GαS43–394

In these hybrids, the prediction is that the structural repeat unit inthe amino terminal domain up to the tetra-leucine motif is 7, and thatswapping sequences in units of 7 will in effect amount to a swap of unitturns of turns of the helical structure that comprises this domain.

A second group of “double crossover” hybrids of this class are thosethat are aligned on the first putative heptad repeat beginning withresidue G11 in GPA1. In these hybrids, helical repeats are swapped fromGPA1 into a GαS backbone one heptad repeat unit at a time.

GαS1-10/GPA11–17/GαS18–394

GαS 1-17/GPA18–24/GαS25–394

GαS1-17/GPA25–31/GαS32–394

GαS?-17/GPA32–38/GαS39–394

The gap that is introduced between residues 9 and 10 in the GαS sequenceis to preserve the alignment of the -LLLLGAGE- (SEQ ID NO:100) sequencemotif. This class of hybrids can be complemented by cassette mutagenesisof each heptad repeat followed by screening of these collections of“heptad” libraries in standard coupling assays.

A third class of hybrids based on the prediction that the amino terminusforms a helical domain with a heptahelical repeat unit are those thateffect the overall hydrophobic or hydrophilic character of the opposingsides of the predicted helical structure (See Lupas et al. supra). Inthis model, the a and d positions of the heptad repeat abcdefg are foundto be conserved hydrophobic residues that define one face of the helix,while the e and g positions define the charged face of the helix. Inthis class of hybrids, the sequence of the GαS parent is maintainedexcept for specific substitutions at one or more of the followingcritical residues to render the different helical faces of GαS more“GPAl-like”

K8Q

+I-10

ElOG

Q12E

R13S

N14D

E15P

E15F

K17L

E21R

K28Q

K32L

V36R

This collection of single mutations could be screened for couplingefficiency to yeast Gβγ and then constructed in combinations (double andgreater if necessary).

A fourth class of hybrid molecules that span this region of GPA1-Gαhybrids are those that have junctions between GPA1 and Gα subunitsintroduced by three primer PCR. In this approach, the two outsideprimers are encoded by sequences at the initiator methionine of GPA1 onthe 5′ side and at the tetraleucine motif of GαS (for example) on the 3′side. A series of junctional primers spanning different junctionalpoints can be mixed with the outside primers to make a series ofmolecules each with different amounts of GPA1 and GαS sequences,respectively.

GPA_(ID) and GPA_(LW) hybrids. The regions of high homology among Gβγsubunits that have been identified by sequence alignment areinterspersed throughout the molecule. The G1 region containing thehighly conserved -GSGESGDST- (SEQ ID NO:2) motif is followed immediatelyby a region of very low sequence consevation, the “il” or insert 1region. Both sequence and length vary considerably among the il regionsof the Gα subunits. By aligning the sequences of Gα subunits, theconserved regions bounding the il region were identified and twoadditional classes of GPA1-Gα hybrids were constructed. The GPA_(ID)hybrids encode the amino terminal 102 residues of GPA1 (up to thesequence -QARKLGIQ-(SEQ ID NO:97) fused in frame to mammalian Gαsubunits, while the GPA_(LW) hybrids encode the amino terminal 244residues of GPA1 (up to the sequence LIHEDIAKA- (SEQ ID NO:3) in GPA1).The reason for constructing the GPA_(ID) and GPA_(LW) hybrids was totest the hypothesis that the il region of GPA1 is required for mediatingthe interaction of GPA1 with yeast Gβγ subunits, for the stableexpression of the hybrid molecules, or for function of the hybridmolecules. The GPA_(ID) hybrids contain the amino terminal domain ofGPA1 fused to the il domain of mammalian subunits, and therefore do notcontain the GPA1 il region, while the GPA_(LW) hybrids contain the aminoterminal 244 residues of GPA1 including the entire il region (as definedby sequence alignments). Hybrids of both GPA_(ID) and GPA_(LW) classeswere constructed for Gαs, C-αi2, Gαi3, Gαo_(a), and Gα16; none of thesehybrids complemented the gpal growth arrest phenotype.

Subsequent to the construction and testing of the GPA_(ID) and GPA_(LW)classes of hybrids, the crystal structures of G_(transducin) in both theGDP and GTPγS-liganded form, and the crystal structure of several Gαilvariants in the GTPγS-liganded and GDP-AlF₄ forms were reported (Noel etal. supra; Lambright et al. supra; and Coleman et al.(1994) Science265:1405–1412). The crystal structures reveal that the ilregion definedby sequence alignment has a conserved structure that is comprised of sixalpha helices in a rigid array, and that the junctions chosen for theconstruction of the GPA_(ID) and GPA_(LW) hybrids were not compatiblewith conservation of the structural features of the il region observedin the crystals. The junction chosen for the GPA_(ID) hybrids falls inthe center of the long αA helix; chimerization of this helix in alllikelihood destabilizes it and the protein structure in general. Thesame is true of the junction chosen for the GPA_(LW) hybrids in whichthe crossover point between GPA1 and the mammalian Gα subunit falls atthe end of the short αC helix and therefore may distort it anddestabilize the protein.

The failure of the GPA_(ID) and GPA_(LW) hybrids is predicted to be dueto disruption of critical structural elements in the il region asdiscussed above. Based upon new alignments and the data presented inNoel et al (supra), Lambright et al (supra), and Coleman et al (supra),this problem can be averted with the ras-like core domain and the ilhelical domain are introduced outside of known structural elements likealpha-helices.

-   -   Hybrid A GαS1-67/GPA66-299/GαS203-394    -   This hybrid contains the entire il insert of GPA1 interposed        into the GαS sequence.    -   Hybrid B GPA1–41/GαS4443-67/GPA66-299/GαS203–394    -   This hybrid contains the amino terminal 41 residues of GPA1 in        place of the 42 amino terminal residues of GαS found in Hybrid        A.        Gαs Hybrids. There is evidence that the “switch region” encoded        by residues 171–237 of Gα transducin (using the numbering of        (Noel et al (supra) also plays a role in Gβγ coupling. First,        the G226A mutation in GαS prevents the GTP-induced        conformational change that occurs with exchange of GDP for GTP        upon receptor activation by ligand. This residue maps to the        highly conserved sequence -DVGGQ- (SEQ ID NO:98), present in all        Gα subunits and is involved in GTP hydrolysis. In both the Gαt        and Gα il crystal structures, this sequence motif resides in the        loop that connects the β3 sheet and the α2 helix in the guanine        nucleotide binding core. In addition to blocking the        conformational change that occurs upon GTP binding, this        mutation also prevents dissociation of GTP-liganded Gαs from        Gβγ. Second, crosslinking data reveals that a highly conserved        cysteine residue in the α2 helix (C215 in Gαo, C210 in Gαt) can        be crosslinked to the carboxy terminal region of Gβ subunits.        Finally, genetic evidence (Whiteway et al. (1993) Mol Cell Biol.        14:3233–3239) identifies an important single residue in GPA1        (E307) in the β2 sheet of the core structure that may be in        direct contact with βγ. A mutation in the GPA1 protein at this        position suppresses the constitutive signalling phenotype of a        variety of STE4 (Gβ) dominant negative mutations that are also        known to be defective in Gα-Gβγ association (as assessed in        two-hybrid assay in yeast as well as by more conventional        genetic tests).

We have tested the hypothesis that there are switch region determinantsinvolved in the association of Gα with Gβγ by constructing a series ofhybrid Gα proteins encoding portions of GPA1 and GαS in differentcombinations.

Two conclusions may be drawn. First, in the context of the aminoterminus of Gαs, the GPA1 switch region suppresses coupling to yeast Gβγ(SGS), while in the context of the GPA1 amino terminus the GPA1 switchregion stabilizes coupling with Gβγ (GPβγ-SGS). This suggests that thesetwo regions of GPA1 collaborate to allow interactions between Gαsubunits and Gβγ subunits. This conclusion is somewhat mitigated by theobservation that the GPA₄₁-Gαs hybrid that does not contain the GPA1switch region is able to complement the growth arrest phenotype of gpalstrains. We have not to date noted a quantitative difference between thebehavior of the GPA₄₁-Gαs allele and the GPA˜I-SGS allele, but if thisinteraction is somewhat degenerate, then it may be difficult toquantitate this accurately. The second conclusion that can be drawn fromthese results is that there are other determinants involved instabilizing the interaction of Gα with Gβγ beyond these two regions asnone of the GPA1/Gαs hybrid proteins couple as efficiently to yeast Gβγas does native GPA1.

The role of the surface-exposed residues of this region may be crucialfor effective coupling to yeast Gβγ, and can be incorporated into hybridmolecules as follows below.

-   -   GαS-GPA-Switch GαS 1-202/GPA298-350/GαS 253-394

This hybrid encodes the entire switch region of GPA 1 in the context ofGαS.

GαS-GPA-α2 GQS 1-2261GPA322-332/GQS 238-394

This hybrid encodes the a² helix of GPA1 in the context of GαS.

GPA41-GαS-GPA-α2GPAl-41/GQS43-226/GPA322-3321GQS238-394

This hybrid encodes the 41 residue amino terminal domain of GPA1 and thea2 helix of GPA1 in the context of GαS.

Finally, the last class of hybrids that will be discussed here are thosethat alter the surface exposed residues of the β2 and β3 sheets of αS sothat they resemble those of the GPA1 QS helix. These altered a2 helicaldomains have the following structure. (The positions of the alteredresidues correspond to GαS.)

L203K

K211E

D215G

K216S

D229S

These single mutations can be engineered into a GαS backbone singly andin pairwise combinations. In addition, they can be introduced in thecontext of both the full length GαS and the GPA₄₁-GαS hybrid describedpreviously. All are predicted to improve the coupling of Gα subunits toyeast Gβγ subunits by virtue of improved electrostatic and hydrophobiccontacts between this region and the regions of Gβ defined by Whitewayand coworkers (Whiteway et al (supra) that define site(s) that interactwith GPA1).

In summary, the identification of hybrid Gα subunits that couple to theyeast pheromone pathway has led to the following general observations.First, all GPA_(BAMH1) hybrids associate with yeast Gβγ, therefore at aminimum these hybrids contain the determinants in GPA1 necessary forcoupling to the pheromone response pathway. Second, the amino terminal41 residues of GPA1 contain sufficient determinants to facilitatecoupling of Gα hybrids to yeast Gβγ in some, but not all, instances, andthat some Gα subunits contain regions outside of the first 41 residuesthat are sufficiently similar to those in GPA1 to facilitate interactionwith GPA1 even in the absence of the amino terminal 41 residues of GPA1.Third, there are other determinants in the first 310 residues of GPA1that are involved in coupling Gα subunits to yeast Gβγ subunits.

The various classes of hybrids noted above are not mutually exclusive.For example, a GPA1 containing GPA1-₄₁ could also feature the L203Kmutation.

While, for the sake of simplicity, we have described hybrids of yeastGPA1 and a mammalian Gαs, it will be appreciated that hybrids may bemade of other yeast Gα subunits and/or other mammalian Gα subunits,notably mammalian Gαi subunits. Moreover, while the described hybridsare constructed from two parental proteins, hybrids of three or moreparental proteins are also possible.

As shown in the Examples, chimeric Gα subunits have been especiallyuseful in coupling receptors to Gαi species.

G. Expression of Gα

Kang et al. supra reported that several classes of native mammalianG˜subunits were able to interact functionally with yeast α subunits whenexpression of Gα was driven from a constitutively active, strongpromoter (PGK) or from a strong inducible promoter (CUP). These authorsreported that rat Gαs, Gαi2 or Gαo expressed at high level coupled toyeast βγ. High level expression of mammalian Gα (i.e. non-stoichiometricwith respect to yeast βγ) is not desirable for uses like those describedin this application. Reconstruction of G protein-coupled receptor signaltransduction in yeast requires the signalling component of theheterotrimeric complex (Gβγ) to be present stoichiometrically with Gαsubunits. An excess of Gα subunits (as was required for coupling ofmammalian Gαi2 and GαO to yeast Gβγ in Kang et al.) would dampen thesignal in systems where Gβγ subunits transduce the signal. An excess ofGα subunits raises the background level of signaling in the system tounacceptably high levels. Preferably, levels of Gα and Gβγ subunits arebalanced. For example, heterologous Gα subunits may be expressed from alow copy (CEN ARS) vector containing the endogenous yeast GPA1 promoterand the GPA1 3′ untranslated region. The minimum criterion, applied to aheterologous Gαsubunit with respect to its ability to couplefunctionally to the yeast pheromone pathway, is that it complement agpal genotype when expressed from the GPA1 promoter on low copy plasmidsor from an integrated, single copy gene. In the work described in thisapplication, all heterologous Gα subunits have been assayed in twobiological systems. In the first assay heterologous Gα subunits aretested for an ability to functionally complement the growth arrestphenotype of gpal strains. In the second assay the transcription of afus1-HIS3 reporter gene is used to measure the extent to which thepheromone response pathway is activated, and hence the extent to whichthe heterologous Gα subunit sequesters the endogenous yeast Gβγ complex.Mammalian Gαs, Gαi2, Gαi3, Gαq, Gα11, Gα16, Gαo_(a), Gαo_(b), and Gαzfrom rat, murine or human origins were expressed from a low copy, CENARS vector containing the GPA1 promoter. Functional complementation ofgpal strains was not observed in either assay system with any of thesefull-length Gα constructs with the exception of rat and human GαS.

H. Chimeric Yeast βγ Subunits

An alternative to the modification of a mammalian Gα subunit forimproved signal transduction is the modification of the pertinent sitesin the yeast Gβ or Gγ subunits. The principles discussed already withrespect to Gα subunits apply, mutatis mutandis, to yeast Gβ or Gγ.

For example, it would not be unreasonable to target the yeast Ste4pGβsubunit with cassette mutagenesis. Specifically, the region of Ste4pthat encodes several of the dominant negative, signaling-defectivemutations would be an excellent target for cassette mutagenesis whenlooking for coupling of yeast Gβγ to specific mammalian Gα subunits.

X. Peptide Libraries

While others have engineered yeast cells to facilitate screening ofexogenous drugs as receptor agonists and antagonists, the cells did notthemselves produce both the drugs and the receptors. Yeast cellsengineered to produce the receptor, but that do not produce the drugsthemselves, are inefficient. To utilize them one must bring a sufficientconcentration of each drug into contact with a number of cells in orderto detect whether or not the drug has an action. Therefore, a microtiterplate well or test tube must be used for each drug. The drug must besynthesized in advance and be sufficiently pure to judge its action onthe yeast cells. When the yeast cell produces the drug, the effectiveconcentration is higher.

Peptide libraries are systems which simultaneously display, in a formwhich permits interaction with a target, a highly diverse and numerouscollection of peptides. These peptides may be presented in solution(Houghten (1992) Biotechniques 13:412–421), or on beads (Lam (1991)Nature 354:82–84), chips (Fodor (1993) Nature 364:555–556), bacteria(Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865–1869) or onphage (Scott and Smith (1990) Science 249:386–390); (Devlin (1990)Science 249:404–406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378–6382); (Felici (1991) J. Mol. Biol. 222:301–310); (Ladnersupra.). Many of these systems are limited in terms of the maximumlength of the peptide or the composition of the peptide (e.g., Cysexcluded). Steric factors, such as the proximity of a support, mayinterfere with binding. Usually, the screening is for binding in vitroto an artificially presented target, not for activation or inhibition ofa cellular signal transduction pathway in a living cell. While a cellsurface receptor may be used as a target, the screening will not revealwhether the binding of the peptide caused an allosteric change in theconformation of the receptor.

The Ladner et al. patent, U.S. Pat. No. 5,096,815, describes a method ofidentifying novel proteins or polypeptides with a desired DNA bindingactivity. Semi-random (“variegated”) DNA encoding a large number ofdifferent potential binding proteins is introduced, in expressible form,into suitable host cells. The target DNA sequence is incorporated into agenetically engineered operon such that the binding of the protein orpolypeptide will prevent expression of a gene product that isdeleterious to the gene under selective conditions. Cells which survivethe selective conditions are thus cells which express a protein whichbinds the target DNA. While it is taught that yeast cells may be usedfor testing, bacterial cells are preferred. The interactions between theprotein and the target DNA occur only in the cell (and then only in thenucleus), not in the periplasm or cytoplasm, and the target is a nucleicacid, and not a receptor protein. Substitution of random peptidesequences for functional domains in cellular proteins permits somedetermination of the specific sequence requirements for theaccomplishment of function. Though the details of the recognitionphenomena which operate in the localization of proteins within cellsremain largely unknown, the constraints on sequence variation ofmitochondrial targeting sequences and protein secretion signal sequenceshave been elucidated using random peptides (Lemire et al., J. Biol.Chem.(1989) 264,20206 and Kaiser et al. (1987) Science 235:312,respectively).

The peptide library of the present invention takes the form of a cellculture, in which essentially each cell expresses one, and usually onlyone, peptide of the library. While the diversity of the library ismaximized if each cell produces a peptide of a different sequence, it isusually prudent to construct the library so there is some redundancy.Depending on size, the combinatorial peptides of the library can beexpressed as is, or can be incorporated into larger fusion proteins. Thefusion protein can provide, for example, stability against degradationor denaturation, as well as a secretion signal if secreted. In anexemplary embodiment of a library for intracellular expression, e.g.,for use in conjunction with intracellular target receptors, thepolypeptide library is expressed as thioredoxin fusion proteins (see,for example, U.S. Pat. Nos. 5,270,181 and 5,292,646; and PCT publicationWO94/02502). The combinatorial peptide can be attached one the terminusof the thioredoxin protein, or, for short peptide libraries, insertedinto the so-called active loop.

In one embodiment, the peptide library is derived to express acombinatorial library of polypeptides which are not based on any knownsequence, nor derived from cDNA. That is, the sequences of the libraryare largely random. In preferred embodiments, the combinatorialpolypeptides are in the range of 3–100 amino acids in length, morepreferably at least 5–50, and even more preferably at least 10, 13, 15,20 or 25 amino acid residues in length. Preferably, the polypeptides ofthe library are of uniform length. It will be understood that the lengthof the combinatorial peptide does not reflect any extraneous sequenceswhich may be present in order to facilitate expression, e.g., such assignal sequences or invariant portions of a fusion protein.

In another embodiment, the peptide library is derived to express acombinatorial library of polypeptides which are based at least in parton a known polypeptide sequence or a portion thereof (not a cDNAlibrary). That is, the sequences of the library is semi-random, beingderived by combinatorial mutagenesis of a known sequence. See, forexample, Ladner et al. PCT publication WO 90/02909; Garrard et al., PCTpublication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007–16010; Griffths et al. (1993) EMBO J. 12:725–734; Clackson etal. (1991) Nature 352:624–628; and Barbas et al. (1992) PNAS89:4457–4461. Accordingly, polypeptide(s) which are known ligands for atarget receptor can be mutagenized by standard techniques to derive avariegated library of polypeptide sequences which can further bescreened for agonists and/or antagonists. For example, the surrogateligand identified for FPRL-1, e.g., theSer-Leu-Leu-Trp-Leu-Thr-Cys-Arg-Pro-Trp-Glu-Ala-Met (SEQ ID NO:4)peptide, can be mutagenized to generate a library of peptides with somerelationship to the original tridecapeptide. This library can beexpressed in a reagent cell of the present invention, and other receptoractivators can be isolated from the library. This may permit theidentification of even more potent FPRL-1 surrogate ligands.

Alternatively, the library can be expressed under conditions wherein thecells are in contact with the original tridecapeptide, e.g., the FPRL-1receptor is being induced by that surrogate ligand. Peptides from anexpressed library can be isolated based on their ability to potentiatethe induction, or to inhibit the induction, caused by the surrogateligand. The latter of course will identify potential antagonists ofchemoattractant receptors. In still other embodiments, the surrogateligand can be used to screen exogenous compound libraries (peptide andnon-peptide) which, by modulating the activity of the identifiedsurrogate, will presumably also similarly effect the native ligand'seffect on the target receptor. In such embodiments, the surrogate ligandcan be applied to the cells, though is preferably produced by thereagent cell, thereby providing an autocrine cell.

In still another embodiment, the combinatorial polypeptides are producedfrom a cDNA library.

In a preferred embodiment of the present invention, the yeast cellscollectively produce a “peptide library”, preferably including at least10³ to 10⁷ different peptides, so that diverse peptides may besimultaneously assayed for the ability to interact with the exogenousreceptor. In an especially preferred embodiment, at least some peptidesof the peptide library are secreted into the periplasm, where they mayinteract with the “extracellular” binding site(s) of an exogenousreceptor. They thus mimic more closely the clinical interaction of drugswith cellular receptors. This embodiment optionally may be furtherimproved (in assays not requiring pheromone secretion) by preventingpheromone secretion, and thereby avoiding competition between thepeptide and the pheromone for signal peptidase and other components ofthe secretion system.

In the present invention, the peptides of the library are encoded by amixture of DNA molecules of different sequence. Each peptide-encodingDNA molecule is ligated with a vector DNA molecule and the resultingrecombinant DNA molecule is introduced into a host cell. Since it is amatter of chance which peptide encoding DNA molecule is introduced intoa particular cell, it is not predictable which peptide that cell willproduce. However, based on a knowledge of the manner in which themixture was prepared, one may make certain statistical predictions aboutthe mixture of peptides in the peptide library.

It is convenient to speak of the peptides of the library as beingcomposed of constant and variable residues. If the nth residue is thesame for all peptides of the library, it is said to be constant. If thenth residue varies, depending on the peptide in question, the residue isa variable one. The peptides of the library will have at least one, andusually more than one, variable residue. A variable residue may varyamong any of two to all twenty of the genetically encoded amino acids;the variable residues of the peptide may vary in the same or differentmanner. Moreover, the frequency of occurrence of the allowed amino acidsat a particular residue position may be the same or different. Thepeptide may also have one or more constant residues.

There are two principal ways in which to prepare the required DNAmixture. In one method, the DNAs are synthesized a base at a time. Whenvariation is desired, at a base position dictated by the Genetic Code, asuitable mixture of nucleotides is reacted with the nascent DNA, ratherthan the pure nucleotide reagent of conventional polynucleotidesynthesis.

The second method provides more exact control over the amino acidvariation. First, trinucleotide reagents are prepared, eachtrinucleotide being a codon of one (and only one) of the amino acids tobe featured in the peptide library. When a particular variable residueis to be synthesized, a mixture is made of the appropriatetrinucleotides and reacted with the nascent DNA. Once the necessary“degenerate” DNA is complete, it must be joined with the DNA sequencesnecessary to assure the expression of the peptide, as discussed in moredetail below, and the complete DNA construct must be introduced into theyeast cell.

XI. Screening and Selection: Assays of Second Messenger Generation

When screening for bioactivity of peptides, intracellular secondmessenger generation can be measured directly. A variety ofintracellular effectors have been identified as beingG-protein-regulated, including adenylyl cyclase, cyclic GMP,phosphodiesterases, phosphoinositidase C, and phospholipase A₂. Inaddition, G proteins interact with a range of ion channels and are ableto inhibit certain voltage-sensitive Ca⁺⁺ transients, as well asstimulating cardiac K⁺ channels.

In one embodiment, the GTPase enzymatic activity by G proteins can bemeasured in plasma membrane preparations by determining the breakdown ofγ³²P GTP using techniques that are known in the art (For example, seeSignal Transduction: A Practical Approach. G. Milligan, Ed. OxfordUniversity Press, Oxford England). When receptors that modulate cAMP aretested, it will be possible to use standard techniques for cAMPdetection, such as competitive assays which quantitate [³H]cAMP in thepresence of unlabelled cAMP.

Certain receptors stimulate the activity of phospholipase C whichstimulates the breakdown of phosphatidylinositol 4,5, bisphosphate to1,4,5-IP3 (which mobilizes intracellular Ca++) and diacylglycerol (DAG)(which activates protein kinase C). Inositol lipids can be extracted andanalyzed using standard lipid extraction techniques. DAG can also bemeasured using thin-layer chromatography. Water soluble derivatives ofall three inositol lipids (IP1, IP2, IP3) can also be quantitated usingradiolabelling techniques or HPLC.

The mobilization of intracellular calcium or the influx of calcium fromoutside the cell can be measured using standard techniques. The choiceof the appropriate calcium indicator, fluorescent, bioluminescent,metallochromic, or Ca++-sensitive microelectrodes depends on the celltype and the magnitude and time constant of the event under study (Borle(1990) Environ Health Perspect 84:45–56). As an exemplary method of Ca++detection, cells could be loaded with the Ca++sensitive fluorescent dyefura-2 or indo-1, using standard methods, and any change in Ca++measured using a fluorometer.

The other product of PIP2 breakdown, DAG can also be produced fromphosphatidyl choline. The breakdown of this phospholipid in response toreceptor-mediated signaling can also be measured using a variety ofradiolabelling techniques.

The activation of phospholipase A2 can easily be quantitated using knowntechniques, including, for example, the generation of arachadonate inthe cell.

In the case of certain receptors, it may be desirable to screen forchanges in cellular phosphorylation. Such assay formats may be usefulwhen the receptor of interest is a receptor tyrosine kinase. Forexample, yeast transformed with the FGF receptor and a ligand whichbinds the FGF receptor could be screened using colony immunoblotting(Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81:7426–7430) usinganti-phosphotyrosine. In addition, tests for phosphorylation could beuseful when a receptor which may not itself be a tyrosine kinase,activates protein kinases that function downstream in the signaltransduction pathway. Likewise, it is noted that protein phosphorylationalso plays a critical role in cascades that serve to amplify signalsgenerated at the receptor. Multi-kinase cascades allow not only signalamplification but also signal divergence to multiple effectors that areoften cell-type specific, allowing a growth factor to stimulate mitosisof one cell and differentiation of another.

One such cascade is the MAP kinase pathway that appears to mediate bothmitogenic, differentiation and stress responses in different cell types.Stimulation of growth factor receptors results in Ras activationfollowed by the sequential activation of c-Raf, MEK, and p44 and p42 MAPkinases (ERK1 and ERK2). Activated MAP kinase then phosphorylates manykey regulatory proteins, including p90RSK and Elk-1 that arephosphorylated when MAP kinase translocates to the nucleus. Homologouspathways exist in mammalian and yeast cells. For instance, an essentialpart of the S. cerevisiae pheromone signaling pathway is comprised of aprotein kinase cascade composed of the products of the STE11, STE7, andFUS3/KSS1 senes (the latter pair are distinct and functionallyredundant). Accordingly, phosphorylation and/or activation of members ofthis kinase cascade can be detected and used to quantitate receptorengagement. Phosphotyrosine specific antibodies are available to measureincreases in tyrosine phosphorylation and phospho-specific antibodiesare commercially available (New England Biolabs, Beverly, Mass.).

Modified methods for detecting receptor-mediated signal transductionexist and one of skill in the art will recognize suitable methods thatmay be used to substitute for the example methods listed.

XII. Screening and Selection Using Reporter Gene Constructs

In addition to measuring second messenger production, reporter geneconstructs can be used. Reporter gene constructs are prepared byoperatively linking a reporter gene with at least one transcriptionalregulatory element. If only one transcriptional regulatory element isincluded it must be a regulatable promoter, At least one the selectedtranscriptional regulatory elements must be indirectly or directlyregulated by the activity of the selected cell-surface receptor wherebyactivity of the receptor can be monitored via transcription of thereporter genes.

The construct may contain additional transcriptional regulatoryelements, such as a FIRE sequence, or other sequence, that is notnecessarily regulated by the cell surface protein, but is selected forits ability to reduce background level transcription or to amplify thetransduced signal and to thereby increase the sensitivity andreliability of the assay.

Many reporter genes and transcriptional regulatory elements are known tothose of skill in the art and others may be identified or synthesized bymethods known to those of skill in the art. Reporter genes

A reporter gene includes any gene that expresses a detectable geneproduct, which may be RNA or protein. Preferred reporter genes are thosethat are readily detectable. The reporter gene may also be included inthe construct in the form of a fusion gene with a gene that includesdesired transcriptional regulatory sequences or exhibits other desirableproperties.

Examples of reporter genes include, but are not limited to CAT(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature282: 864–869) luciferase, and other enzyme detection systems, such asbeta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell.Biol. 7:725–737); bacterial luciferase (Engebrecht and Silverman (1984),PNAS 1: 4154–4158; Baldwin et al. (1984), Biochemistry 23: 3663–3667);alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231–238,Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secretedalkaline phosphatase (Cullen and Malim (1992) Methods in Enzymol.216:362–368).

Transcriptional control elements include, but are not limited to,promoters, enhancers, and repressor and activator binding sites.Suitable transcriptional regulatory elements may be derived from thetranscriptional regulatory regions of genes whose expression is rapidlyinduced, generally within minutes, of contact between the cell surfaceprotein and the effector protein that modulates the activity of the cellsurface protein. Examples of such genes include, but are not limited to,the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477–485),such as c-fos, Immediate early genes are genes that are rapidly inducedupon binding of a ligand to a cell surface protein. The transcriptionalcontrol elements that are preferred for use in the gene constructsinclude transcriptional control elements from immediate early genes,elements derived from other genes that exhibit some or all of thecharacteristics of the immediate early genes, or synthetic elements thatare constructed such that genes in operative linkage therewith exhibitsuch characteristics. The characteristics of preferred genes from whichthe transcriptional control elements are derived include, but are notlimited to, low or undetectable expression in quiescent cells, rapidinduction at the transcriptional level within minutes of extracellularsimulation, induction that is transient and independent of new proteinsynthesis, subsequent shut-off of transcription requires new proteinsynthesis, and mRNAs transcribed from these genes have a shorthalf-life. It is not necessary for all of these properties to bepresent.

In the most preferred constructs, the transcriptional regulatoryelements are derived from the c-fos gene.

The c-fos proto oncogene is the cellular homolog of the transforminggene of FBJ osteosarcoma virus. It encodes a nuclear protein that mostlikely involved in normal cellular growth and differentiation.Transcription of c-fos is transiently and rapidly activated by growthfactors and by other inducers of other cell surface proteins, includinghormones, differentiation-specific agents, stress, mitogens and otherknown inducers of cell surface proteins. Activation is protein synthesisindependent. The c-fos regulatory elements include (see, Verma et al.(1987) Cell 51: a TATA box that is required for transcriptioninitiation; two upstream elements for basal transcription, and anenhancer, which includes an element with dyad symmetry and which isrequired for induction by TPA, serum, EGF, and PMA.

The 20 bp transcriptional enhancer element located between −317 and −298bp upstream from the c-fos mRNA cap site, which is essential for seruminduction in serum starved NIH 3T3 cells. One of the two upstreamelements is located at −63—57 and it resembles the consensus sequencefor cAMP regulation.

Other promoters and transcriptional control elements, in addition tothose described above, include the vasoactive intestinal peptide (VIP)gene promoter (cAMP responsive; Fink et al. (1988), Proc. Natl. Acad.Sci. 85:6662–6666); the somatostatin gene promoter (cAMP responsive;Montminy et al. (1986), Proc. Natl. Acad. Sci. 8.3:6682–6686); theproenkephalin promoter (responsive to cAMP, nicotinic agonists, andphorbol esters; Comb et al. (1986), Nature 323:353–356); thephosphoenolpyruvate carboxy-kinase gene promoter (cAMP responsive; Shortet al. (1986), J. Biol. Chem. 261:9721–9726); the NGFI-A gene promoter(responsive to NGF, cAMP, and serum; Changelian et al. (1989). Proc.Natl. Acad. Sci. 86:377–381); and others that may be known to orprepared by those of skill in the art.

In certain assays it may be desirable to use changes in growth in thescreening procedure. For example, one of the consequences of activationof the pheromone signal pathway in wild-type yeast is growth arrest. Ifone is testing for an antagonist of a G protein-coupled receptor, thisnormal response of growth arrest can be used to select cells in whichthe pheromone response pathway is inhibited. That is, cells exposed toboth a known agonist and a peptide of unknown activity will be growtharrested if the peptide is neutral or an agonist, but will grow normallyif the peptide is an antagonist. Thus, the growth arrest response can beused to advantage to discover peptides that function as antagonists.

However, when searching for peptides which can function as agonists of Gprotein-coupled receptors, or other pheromone system proteins, thegrowth arrest consequent to activation of the pheromone response pathwayis an undesirable effect since cells that bind peptide agonists stopgrowing while surrounding cells that fail to bind peptides will continueto grow. The cells of interest, then, will be overgrown or theirdetection obscured by the background cells, confounding identificationof the cells of interest. To overcome this problem the present inventionteaches engineering the cell such that: 1) growth arrest does not occuras a result of exogenous signal pathway activation (e.g., byinactivating the FAR1 gene); and/or 2) a selective growth advantage isconferred by activating the pathway (e.g., by transforming anauxotrophic mutant with a HIS3 gene under the control of apheromone-responsive promoter, and applying selective conditions).

It is, of course, desirable that the exogenous receptor be exposed on acontinuing basis to the peptides. Unfortunately, this is likely toresult in desensitization of the pheromone pathway to the stimulus. Forexample, the mating signal transduction pathway is known to becomedesensitized by several mechanisms including pheromone degradation andmodification of the function of the receptor, G proteins,s and/ordownstream elements of the pheromone signal transduction by the productsof the SST2, STE50, AFR1 (Konopka, J. B. (1993) Mol. Cell. Biol.13:6876–6888) and SGV1, MSG5, and SIG1 genes. Selected mutations inthese genes can lead to hypersensitivity to pheromone and an inabilityto adapt to the presence of pheromone. For example, introduction ofmutations that interfere with function into strains expressingheterologous G protein-coupled receptors constitutes a significantimprovement on wild type strains and enables the development ofextremely sensitive bioassays for compounds that interact with thereceptors. Other mutations e.g. STE50, sgv1, bar1, ste2, ste3, pik1,msg5, sig1, and aft1, have the similar effect of increasing thesensitivity of the bioassay. Thus desensitization may be avoided bymutating (which may include deleting) the SST2 gene so that it no longerproduces a functional protein, or by mutating one of the other geneslisted above.

If the endogenous homolog of the receptor is produced by the yeast cell,the assay will not be able to distinguish between peptides whichinteract with the endogenous receptor and those which interact with theexogenous receptor. It is therefore desirable that the endogenous genebe deleted or otherwise rendered nonfunctional.

In the case of receptors which modulate cyclic AMP, a transcriptionalbased readout can be constructed using the cyclic AMP response elementbinding protein, CREB, which is a transcription factor whose activity isregulated by phosphorylation at a particular serine (S133). When thisserine residue is phosphorylated, CREB binds to a recognition sequenceknown as a CRE (cAMP Responsive Element) found to the 5′ of promotorsknown to be responsive to elevated cAMP levels. Upon binding ofphosphorylated CREB to a CRE, transcription from this promoter isincreased.

Phosphorylation of CREB is seen in response to both increased cAMPlevels and increased intracellular Ca levels. Increased cAMP levelsresult in activation of PKA, which in turn phosphorylates CREB and leadsto binding to CRE and transcriptional activation. Increasedintracellular calcium levels results in activation of calcium/calmodulinresponsive kinase IV (CaM kinase IV). Phosphorylation of CREB by CaMkinase IV is effectively the same as phosphorylation of CREB by PKA, andresults in transcriptional activation of CRE containing promotors.

Therefore, a transcriptional-based readout can be constructed in cellscontaining a reporter gene whose expression is driven by a basalpromoter containing one or more CRE. Changes in the intracellularconcentration of Ca⁺⁺ (a result of alterations in the activity of thereceptor upon engagement with a ligand) will result in changes in thelevel of expression of the reporter gene if: a) CREB is alsoco-expressed in the cell, and b) either the endogenous yeast CaM kinasewill phosphorylate CREB in response to increases in calcium or if anexogenously expressed CaM kinase IV is present in the same cell. Inother words, stimulation of PLC activity will result in phosphorylationof CREB and increased transcription from the CRE-construct, whileinhibition of PLC activity will result in decreased transcription fromthe CRE-responsive construct.

As described in Bonni et al. (1993) Science 262:1575–1579, theobservation that CNTF treatment of SK-N-MC cells leads to the enhancedinteraction of STAT/p91 and STAT related proteins with specific DNAsequences suggested that these proteins might be key regulators ofchanges in gene expression that are triggered by CNTF. Consistent withthis possibility is the finding that DNA sequence elements similar tothe consensus DNA sequence required for STAT/p91 binding are presentupstream of a number of genes previously found to be induced by CNTF(e.g., Human c-fos, Mouse c-fos, Mouse tis11, Rat junB, Rat SOD-1, andCNTF). Those authors demonstrated the ability of STAT/p91 binding sitesto confer CNTF responsiveness to a non-responsive reporter gene.Accordingly, a reporter construct for use in the present invention fordetecting signal transduction through STAT proteins, such as fromcytokine receptors, can be generated by using −71 to +109 of the mousec-fos gene fused to the bacterial chloramphenicol acetyltransferase gene(−71fosCAT) or other detectable marker gene. Induction by a cytokinereceptor induces the tyrosine phosphorylation of STAT and STAT-relatedproteins, with subsequent translocation and binding of these proteins tothe STAT-RE. This then leads to activation of transcription of genescontaining this DNA element within their promoters.

In preferred embodiments, the reporter gene is a gene whose expressioncauses a phenotypic change which is screenable or selectable. If thechange is selectable, the phenotypic change creates a difference in thegrowth or survival rate between cells which express the reporter geneand those which do not. If the change is screenable, the phenotypechange creates a difference in some detectable characteristic of thecells, by which the cells which express the marker may be distinguishedfrom those which do not. Selection is preferable to screening in that itcan provide a means for amplifying from the cell culture those cellswhich express a test polypeptide which is a receptor effector.

The marker gene is coupled to the receptor signaling pathway so thatexpression of the marker gene is dependent on activation of thereceptor. This coupling may be achieved by operably linking the markergene to a receptor-responsive promoter. The term “receptor-responsivepromoter” indicates a promoter which is regulated by some product of thetarget receptor's signal transduction pathway.

Alternatively, the promoter may be one which is repressed by thereceptor pathway, thereby preventing expression of a product which isdeleterious to the cell. With a receptor repressed promoter, one screensfor agonists by linking the promoter to a deleterious gene, and forantagonists, by linking it to a beneficial gene. Repression may beachieved by operably linking a receptor-induced promoter to a geneencoding mRNA which is antisense to at least a portion of the mRNAencoded by the marker gene (whether in the coding or flanking regions),so as to inhibit translation of that mRNA. Repression may also beobtained by linking a receptor-induced promoter to a gene encoding a DNAbinding repressor protein, and incorporating a suitable operator siteinto the promoter or other suitable region of the marker gene.

In the case of yeast, suitable positively selectable (beneficial) genesinclude the following: URA3, LYS2, HIS3, LEU2, TRP1; ADE1,2,3,4,5, 7,8;ARGI, 3, 4, 5, 6, 8; HIS1, 4, 5; ILV1, 2, 5; THR1, 4; TRP2, 3, 4, 5;LEU1,4; MET2,3,4,8,9,14,16,19; URA1,2,4,5,10; H0M3,6; ASP3; CHO1; ARO 2,7; CYS3; OLE1; IN01,2,4; PR01, 3 Countless other genes are potentialselective markers. The above are involved in well-characterizedbiosynthetic pathways. The imidazoleglycerol phosphate dehydratase (IGPdehydratase) gene (HIS3) is preferred because it is both quite sensitiveand can be selected over a broad range of expression levels. In thesimplest case, the cell is auxotrophic for histidine (requires histidinefor growth) in the absence of activation. Activation leads to synthesisof the enzyme and the cell becomes prototrophic for histidine (does notrequire histidine). Thus the selection is for growth in the absence ofhistidine. Since only a few molecules per cell of IGP dehydratase arerequired for histidine prototrophy, the assay is very sensitive.

In a more complex version of the assay, cells can be selected forresistance to aminotriazole (AT), a drug that inhibits the activity ofIGP dehydratase. Cells with low, fixed level of expression of HIS3 aresensitive to the drug, while cells with higher levels are resistant. Theamount of AT can be selected to inhibit cells with a basal level of HIS3expression (whatever that level is) but allow growth of cells with aninduced level of expression. In this case selection is for growth in theabsence of histidine and in the presence of a suitable level of AT.

In appropriate assays, so-called counterselectable or negativelyselectable genes may be used. Suitable genes include: URA3(orotidine-5′-phosphate decarboxylase; inhibits growth on 5-fluorooroticacid), LYS2 (2-aminoadipate reductase; inhibits growth on α-aminoadipateas sole nitrogen source), CYH2 (encodes ribosomal protein L29;cycloheximide-sensitive allele is dominant to resistant allele), CAN1(encodes arginine penmease; null allele confers resistance to thearginine analog canavanin), and other recessive drug-resistant markers.

In one example, the marker gene effects yeast cell growth. The naturalresponse to signal transduction via the yeast pheromone system responsepathway is for cells to undergo growth arrest. This is the preferred wayto select for antagonists to a ligand/receptor pair that induces thepathway. An autocrine peptide antagonist would inhibit the activation ofthe pathway; hence, the cell would be able to grow. Thus, the FAR1 genemay be considered an endogenous counterselectable marker. The FAR1 geneis preferably inactivated when screening for agonist activity.

The marker gene may also be a screenable gene. The screenedcharacteristic may be a change in cell morphology, metabolism or otherscreenable features. Suitable markers include beta-galactosidase (Xgal,C₁₂FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)), alkalinephosphatase, horseradish peroxidase, exo-glucanase (product of yeastexbl gene; nonessential, secreted); luciferase; bacterial greenfluorescent protein; (human placental) secreted alkaline phosphatase(SEAP); and chloramphenicol transferase (CAT). Some of the above can beengineered so that they are secreted (although not β-galactosidase). Apreferred screenable marker gene is beta-galactosidase; yeast cellsexpressing the enzyme convert the colorless substrate Xgal into a bluepigment. Again, the promoter may be receptor-induced orreceptor-inhibited.

XIII. Genetic Markers in Yeast Strains

Yeast strains that are auxotrophic for histidine (HIS3) are known, seeStruhl and Hill, (1987) Mol. Cell. Biol., 7:104; Fasullo and Davis, Mol.Cell. Biol., (1988) 8:4370. The HIS3 (imidazoleglycerol phosphatedehydratase) gene has been used as a selective marker in yeast. SeeSikorski and Heiter, (1989) Genetics, 122:19; Struhl, et al., P.N.A.S.(1979) 76:1035; and, for FUS1-HIS3 fusions, see Stevenson, et al.,(1992) Genes Dev., 6:1293.

IXX. Novel FPRL-1 Ligand

Yet another aspect of the invention pertains to a novel ligand for theorphan receptor, FPRL-1. As described in Example 8, a tridecapeptidehaving the sequence Ser-Leu-Leu-Trp-Leu-Thr-Cys-Arg-Pro-Trp-Glu-Ala-Met(SEQ ID NO:4) was identified from a polypeptide library on the basis ofits ability to act as a surrogate ligand for FPRL-1.

Chemoattractants are important mediators of inflammation, they functionto recruit phagocytic cells at the site of injury or infection. Theyalso mediate granule secretion, superoxide generation and upregulationof cell surface adhestion molecules, for example MAC-1. Exemplarychemoattractants include the complement component C5a, interleukin 8,leukotriene B4 and platelet activiating factor. Many of these substancesparticipate in pathophysiological conditions such as anaphylaxis andseptic shock. The identification of ligands for the orphan FPRL1receptor provides new opportunities for discovery of receptor agonists,that could potentially serve to enhance lymphocyte recruitment inimmunocompromised patients, and for the discovery of receptorantagonists (described supra) that could prevent undesirableconsequences of immune activation such as anaphylactic or septic shock.

The term “peptide” is used herein to refer to a chain of two or moreamino acids or amino acid analogs (including non-naturally occurringamino acids), with adjacent amino acids joined by peptide (—NHCO—)bonds. Thus, the peptides of the present invention includeoligopeptides, polypeptides, and proteins. Preferably, the peptides ofthe present invention include all or a portion of theS-L-L-W-L-T-C-R-P-W-E-A-M (SEQ ID NO:4) peptide, or a homolog thereof.The peptide (or peptidomimetic) is preferably at least 3 amino acidresidues in length, though peptides of 4, 5, 7, 10, 13 or more residuesin length are contemplated. For example, the sequence derived from theFPRL-1 surrogate ligand can be provided as part of a fusion protein. Theminimum peptide length is chiefly dictated by the need to obtainsufficient potency as an activator or inhibitor. Given the size of thepeptide isolated in subject assay, smaller fragments of thetridecapeptide which retain receptor binding activity will be easilyidentified, e.g., by chemical synthesis of different fragments. Themaximum peptide length will only be a function of synthetic convenienceonce an active peptide is identified.

The invention also provides for the generation of mimetics, e.g. peptideor non-peptide agents. Moreover, the present invention also contemplatesvariants of the subject polypeptide which may themselves be eitheragonistic or antagonistic of the S-L-L-W-L-T-C—R-P-W-E-A-M (SEQ ID NO:4)peptide. Thus, using such mutagenic techniques as known in the art, thedeterminants of S-L-L-W-L-T-C-R-P-W-E-A-M (SEQ ID NO:4) polypeptidewhich participate in FPRL-1 interactions can be ellucidated. Toillustrate, the critical residues of a subject polypeptide which areinvolved in molecular recognition of an FPRL-1 receptor can bedetermined and used to generate variant polypeptides which competitivelyinhibit binding of the authentic S-L-L-W-L-T-C-R-P-W-E-A-M (SEQ ID NO:4)peptide with that receptor. By employing, for example, scanningmutagenesis to map the amino acid residues of the polypeptide involvedin binding the FPRL-1 receptor, peptide and peptidomimetic compounds canbe generated which mimic those residues in binding to the receptor andwhich consequently can inhibit binding of an authentic ligand for theFPRL-1 receptor and interfere with the function of that receptor.

Moreover, as is apparent from the present and parent disclosures,mimetopes of the subject S-L-L-W-L-T-C-R-P-W-E-A-M (SEQ ID NO:4) peptidecan be provided as non-hydrolyzable peptide analogs. For illustrativepurposes, peptide analogs of the present invention can be generatedusing, for example, benzodiazepines (e.g., see Freidinger et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al.in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988, p123), C-7 mimics (Huffman et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptides(Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. inPeptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (NaGαi et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), β-aminoalcohols (Gordonet al. (1985) Biochem Biophys Res Commun126:419; and Dann et al. (1986)Biochem Biophys Res Commun 134:71), diaminoketones (Natarajan et al.(1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modifed(Roark et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988, p134). Also, see generally,Session III: Analytic and synthetic methods, in in Peptides: Chemistryand Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988) In an exemplary embodiment, the peptidomimetic can be derived as aretro-inverso analog of the peptide. To illustrate, theS-L-L-W-L-T-C-R-P-W-E-A-M (SEQ ID NO:4) peptide can be generated as theretro-inverso analog:

Such retro-inverso analogs can be made according to the methods known inthe art, such as that described by the Sisto et al. U.S. Pat. No.4,522,752. For example, the illustrated retro-inverso analog can begenerated as follows. The geminal diamine corresponding to the serineanalog is synthesized by treating a protected serine with ammonia underHOBT-DCC coupling conditions to yield the N-Boc amide, and theneffecting a Hofmann-type rearrangement withI,I-bis-(trifluoroacetoxy)iodobenzene (TIB), as described inRadhakrishna et al. (1979) J. Org. Chem. 44:1746. The product amine saltis then coupled to a side-chain protected (e.g., as the benzyl ester)N-Fmoc D-Leu residue under standard conditions to yield thepseudodipeptide. The Fmoc (fluorenylmethoxycarbonyl) group is removedwith piperidine in dimethylformamide, and the resulting amine istrimethylsilylated with bistrimethylsilylacetamide (BSA) beforecondensation with suitably alkylated, side-chain protected derivative ofMeldrum's acid, as described in U.S. Pat. No. 5,061,811 to Pinori etal., to yield the retro-inverso tripeptide analog S-L-L. Thepseudotripeptide is then coupled with L-Trp under standard conditions togive the protected tetrapeptide analog. The protecting groups areremoved to release the product, and the steps repeated to enlogate thetetrapeptide to the full length peptide. It will be understood that amixed peptide, e.g. including some normal peptide linkages, can begenerated. As a general guide, sites which are most susceptible toproteolysis are typically altered, with less susceptible amide linkagesbeing optional for mimetic switching The final product, or intermediatesthereof, can be purified by HPLC.

In another illustrative embodiment, the peptidomimetic can be derived asa retro-enatio analog of the peptide, such as the exemplary retro-enatiopeptide analog derived for the illustrative S-L-L-W-L-T-C-R-P-W-E-A-M(SEQ ID NO:4) peptide:

NH₃-(d) Met-(d) Ala-(d) Glu-(d)Trp . . . (d) Trp-(d) Leu-(d)-Leu-(d) Ser

Retro-enantio analogs such as this can be synthesized using commerciallyavailable D-amino acids and standard solid- or solution-phasepeptide-synthesis techniques. For example, in a preferred solid-phasesynthesis method, a suitably amino-protected (t-butyloxycarbonyl, Boc)D-Serine residue (or analog thereof) is covalently bound to a solidsupport such as chloromethyl resin. The resin is washed withdichloromethane (DCM), and the BOC protecting group removed by treatmentwith TFA in DCM. The resin is washed and neutralized, and the nextBoc-protected D-amino acid (D-Leu) is introduced by coupling withdiisopropylcarbodiimide. The resin is again washed, and the cyclerepeated for each of the remaining amino acids in turn (D-Leu, D-Trpetc). When synthesis of the protected retro-enantio peptide is complete,the protecting groups are removed and the peptide cleaved from the solidsupport by treatment with hydrofluoric acid/anisole/dimethylsulfide/thioanisole. The final product is purified by HPLC to yield thepure retro-enantio analog.

In still another illustrative embodiment, trans-olefin derivatives canbe made for the subject polypeptide. For example, an exemplary olefinanalog is derived for the illustrative S-L-L-W-L-T-C-R-P-W-E-A-M (SEQ IDNO:4) peptide:

The trans olefin analog of the subject peptide can be synthesizedaccording to the method of Y. K. Shue et al. (1987) Tetrahedron Letters28:3225.

Still another class of peptidomimetic derivatives include thephosphonate derivatives, such as the partially phosphonate derivativedS-L-L-W-L-T-C-R-P-W-E-A-M (SEQ ID NO:4) peptide:

The synthesis of such phosphonate derivatives can be adapted from knownsynthesis schemes. See, for example, Loots et al. in Peptides: Chemistryand Biology, (Escom Science Publishers, Leiden, 1988, p. 118); Petrilloet al. in Peptides: Structure and Function (Proceedings of the 9thAmerican Peptide Symposium, Pierce Chemical Co. Rockland, Ill., 1985).

EXEMPLIFICATION

The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention and are not intended to limit the invention.

Example 1 Development of Autocrine Yeast Strains

In this example, we describe a pilot experiment in which haploid cellswere engineered to be responsive to their own pheromones. (Note that inthe examples, functional genes are capitalized and inactivated genes arein lower case.) For this purpose we constructed recombinant DNAmolecules designed to:

i. place the coding region of STE2 under the transcriptional control ofelements which normally direct the transcription of STE3. This is donein a plasmid that allows the replacement of genomic STE3 of S.cerevisiae with sequences wherein the coding sequence of STE2 is drivenby STE3 transcriptional control elements.

ii. place the coding region of STE3 under the transcriptional control ofelements which normally direct the transcription of STE2. This is donein a plasmid which will allow the replacement of genomic STE2 of S.cerevisiae with sequences wherein the coding sequence of STE3 is drivenby STE2 transcriptional control elements.

The sequence of the STE2 gene is known see Burkholder A. C. and HartwellL. H. (1985), Nuc. Acids Res. 13, 8463; Nakayama N., Miyajima A., AraiK. (1985) EMBO J. 4, 2643.

A 4.3 kb BamHI fragment that contains the entire STE2 gene was excisedfrom plasmid YEp24-STE2 (obtained from J. Thorner, Univ. of California)and cloned into pALTER (Protocols and Applications Guide, 1991, PromegaCorporation, Madison, Wis.). An SpeI site was introduced 7 nucleotides(nts) upstream of the ATG of STE2 with the following mutagenicoligonucleotide, using the STE2 minus strand as template:

5′-GTTAAGAACCATATACTAGTATCAAAAATGTCTG 3′ (SEQ ID NO:5)

A second SpeI site was simultaneously introduced just downstream of theSTE2 stop codon with the following mutagenic oligonucleotide:

5′-TGATCAAAATTTACTAGTTTGAAAAAGTAATTTCG 3′ (SEQ ID NO:6)

The BamHI fragment of the resulting plasmid (Cadus 1096) containing STE2with SpeI sites immediately flanking the coding region, was thensubcloned into the yeast integrating vector YIpl9 to yield Cadus 1143.

The STE3 sequence is also known (Nakayama N., Miyajima A., Arai K.(1985), EMBO J. 4, 2643; (Hagen D. C., McCaffrey G., Sprague G. F.(1986), Proc. Natl. Acad. Sci. 83, 1418. STE3 was made available by Dr.J. Broach as a 3.1 kb fragment cloned into pBLUESCRIPT-KS II(Stratagene, 11011 North Torrey Pines Road, La Jolla, Calif. 92037).STE3 was subcloned as a KpnI-XbaI fragment into both M13 mpl8 RF (toyield Cadus 1105 and pUC19 (to yield Cadus 1107). The two SpeI sites inCadus 1107 were removed by digestion with SpeI, fill-in with DNApolymerase I Klenow fragment, and recircularization by blunt-endligation. Single-stranded DNA containing the minus strand of STE3 wasobtained using Cadus 1105 and SpeI sites were introduced 9 nts upstreamof the start codon and 3 nts downstream of the stop codon of STE3 withthe following mutagenic oligonucleotides, respectively:

5′-GGCAAAATACTAGTAAAATTTTCATGTC 3′ (SEQ ID NO:7) 5′- (3′ SEQ ID NO:8)GGCCCTTAACACACTAGTGTCGCATTATATTTAC

The mutagenesis was accomplished using the T7-GEN protocol of UnitedStates Biochemical (T7-GEN In Vitro Mutagenesis Kit, Descriptions andProtocols, 1991, United States Biochemical, P.O. Box 22400, Cleveland,Ohio 44122). The replicative form of the resulting Cadus 1141 wasdigested with AflII and KpnI, and the approximately 2 kb fragmentcontaining the entire coding region of STE3 flanked by the two newlyintroduced Spe I sites was isolated and ligated with the approximately3.7 kb vector fragment of AflII- and KpnI-digested Cadus 1107, to yieldCadus 1138. Cadus 1138 was then digested with XbaI and KpnI, and theSTE3-containing 2.8 kb fragment was ligated into the XbaI- andKpnI-digested yeast integrating plasmid pRS406 (Sikorski, R. S. andHieter, P. (1989), Genetics 122:19–27) to yield Cadus 1145.

The SpeI fragment of Cadus 1143 was replaced with the SpeI fragment ofCadus 1145 to yield Cadus 1147, in which the coding sequences of STE3are under the control of STE2 expression elements. Similarly, the SpeIfragment of Cadus 1145 was replaced with the SpeI fragment of Cadus 1143to yield Cadus 1148, in which the coding sequences of STE2 are under thecontrol of STE3 expression elements. Using the method of pop-in/pop-outreplacement (Rothstein, R. (1991) Methods in Enzymology, 194:281 301),Cadus 1147 was used to replace genomic STE2 with the ste2-STE3 hybrid ina MATa cell and Cadus 1148 was used to replace genomic STE3 with theste3-STE2 hybrid in a MATa cell. Cadus 1147 and 1148 contain theselectable marker URA3.

Haploid yeast of mating type a which had been engineered to express HIS3under the control of the pheromone-inducible FUS1 promoter weretransformed with CADUS 1147, and transformants expressing URA3 wereselected. These transformants, which express both Ste2p and Ste3p, wereplated on 5-fluoroorotic acid to allow the selection of clones which hadlost the endogenous STE2, leaving in its place the heterologous,integrated STE3. Such cells exhibited the ability to grow on mediadeficient in histidine, indicating autocrine stimulation of thepheromone response pathway.

Similarly, haploids of mating type α that can express HIS3 under thecontrol of the pheromone-inducible FUS1 promoter were transformed withCADUS 1148 and selected for replacement of their endogenous STE3 withthe integrated STE2. Such cells showed, by their ability to grow onhistidine-deficient media, autocrine stimulation of the pheromoneresponse pathway.

Example 2 Strain Development

In this example, yeast strains are constructed which will facilitateselection of clones which exhibit autocrine activation of the pheromoneresponse pathway. To construct appropriate yeast strains, we will use:the YIp-STE3 and pRS-STE2 knockout plasmids described above, plasmidsavailable for the knockout of FAR1, SST2, and HIS3, and mutant strainsthat are commonly available in the research community. The followinghaploid strains will be constructed, using one-step or two-step knockoutprotocols described in Meth. Enzymol 194:281–301, 1991:

1. MATα ste3::STE2::ste3 far1 sst2 FUS1::HIS3 2. MATαste2::STE3::ste2 far1 sst2 FUS1::HIS3 3. MATαste3::STE2::ste3 far1 sst2 mfα1 mfα2 FUS1::HIS3 4. MATa ste2::STE3::ste2 far1 sst2 mfa1  mfa2  FUS1::HIS3 5. MATa  bar1 far1-1fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3 6. MATa mfa1 mfa2 far1-1his3::fus1-HIS3 ste2-STE3 ura3 met1 ade1 leu2

Strains 1 and 2 will be tested for their ability to grow onhistidine-deficient media as a result of autocrine stimulation of theirpheromone response pathways by the pheromones which they secrete. Ifthese tests prove successful, strain 1 will be modified to inactivateendogenous MFα1 and MFα2. The resulting strain 3, MATα far1 sst2ste3::STE2::ste3 FUS1::HIS3 mfal mfa2, should no longer display theselectable phenotype (i.e., the strain should be auxotrophic forhistidine). Similarly, strain 2 will be modified to inactivateendogenous MFa1 and MFa2. The resulting strain 4, MATa far1 sst2ste2::STE3::ste2 FUS1::HIS3 Mfal mfa2, should be auxotrophic forhistidine. The uses of strains 5 and 6 are outlined in Examples 3 and 4below.

Example 3 Peptide Library

In this example, a synthetic oligonucleotide encoding a peptide isexpressed so that the peptide is secreted or transported into theperiplasm.

i. The region of MFα1 which encodes mature α-factor has been replacedvia single-stranded mutagenesis with restriction sites that can acceptoligonucleotides with AflII and BglII ends. Insertion ofoligonucleotides with AflII and BglII ends will yield plasmids whichencode proteins containing the MFα1 signal and leader sequences upstreamof the sequence encoded by the oligonucleotides. The MFα1 signal andleader sequences should direct the processing of these precursorproteins through the pathway normally used for the transport of matureα-factor.

The MFα1 gene, obtained as a 1.8 kb EcoRI fragment from pDA6300 (J.Thorner, Univ. of California) was cloned into pALTER in preparation foroligonucleotide-directed mutagenesis to remove the coding region ofmature α-factor while constructing sites for acceptance ofoligonucleotides with AflII and BclI ends. The mutagenesis wasaccomplished using the minus strand as template and the followingmutagenic oligonucleotide:

5′-CTAAAGAAGAAGGGGTATCTTTGCTTAAGCTCGAGATCTCGACTGATA-ACAACAGTGTAG-3′ (SEQID NO:9)

A HindIII site was simultaneously introduced 7 nts upstream of the MFα1start codon with the oligonucleotide:

5′CATACACAATATAAAGCTTTAAAAGAATGAG-3′ (SEQ ID NO:10)

The resulting plasmid, Cadus 1214, contains a HindIII site 7 ntsupstream of the MFα 1 initiation codon, an AflII site at the positionswhich encode the KEX2 processing site in the MFα1 leader peptide, andXhoI and BglII sites in place of all sequences from the leader-encodingsequences up to and including the normal stop codon. The 1.5 kb HindIIIfragment of Cadus 1214 therefore provides a cloning site foroligonucleotides to be expressed in yeast and secreted through thepathway normally travelled by endogenous α-factor.

A sequence comprising the ADC1 promoter and 5′ flanking sequence wasobtained as a 1.5 kb BamHI-HindIII fragment from pAAH5 (Ammerer, G.(1983) Academic Press, Inc., Meth. Enzymol. 101, 192–201 and ligatedinto the high copy yeast plasmid pRS426 (Christianson, T. W et al.(1992) Gene 110:119–122) (see FIG. 1). The unique XhoI site in theresulting plasmid was eliminated to yield Cadus 1186. The 1.5 Kb HindIIIfragment of Cadus 1214 was inserted into HindIII-digested Cadus 1186;expression of sequences cloned into this cassette initiates from theADH1 promoter. The resulting plasmid, designated Cadus 1215, can beprepared to accept oligonucleotides with AflII and BclI ends bydigestion with those restriction endonucleases. The oligonucleotideswill be expressed in the context of MF α1 signal and leader peptides(FIG. 2).

Modified versions of Cadus 1215 were also constructed. To 30 improve theefficiency of ligation of oligonucleotides into the expression vector,Cadus 1215 was restricted with KpnI and religated to yield Cadus 1337.This resulted in removal of one of two HindIII sites. Cadus 1337 waslinearized with HindIII, filled-in, and recircularized to generate Cadus1338. To further tailor the vector for library construction, thefollowing double-stranded oligonucleotide was cloned into AflII-andBglII-digested Cadus 1338:

5′ TTAAGCGTGAGGCAGAAGCTTATCGATA oligo 062 (SEQ ID NO:11) 3′CGCACTCCGTCTTCGAATAGCTATCTAG oligo 063 (SEQ ID NO:12)

The ClaI site is unique in the resulting vector, Cadus 1373. In Cadus1373, the HindIII site that exists at the junction between the MFα prosequence and the mature peptide to be expressed by this vector was madeunique. Therefore the HindIII site and the downstream BglII site can beused to insert oligo-nucleotides encoding peptides of interest. Thesemodifications of Cadus 1215 provide an laternative to the use of theAflII site in the cloning of oligonucleotides into the expressionsvector.

Cadus 1373 was altered further to permit elimination from restrictedvector preparations of contaminating singly-cut plasmid. Suchcontamination could result in unacceptably high backgroundtransformation. To eliminate this possibility, approximately 1.1 kb ofdispensable ADH1 sequence at the 5′ side of the promoter region wasdeleted. This was accomplished by restruction of Cadus 1373 with SphIand BamHI, fill-in, and ligation; this maneuver regenerates the BamHIsite. The resulting vector, Cadus 1624, was then restricted with HindIIIand ClaI and an approximately 1.4 kb HindIII and ClaI fragment encoding25 lacZ was inserted to generate Cadus 1625. Use of HindIII- andBglII-restricted Cadus 1625 for acceptance of oligonucleotides resultsin a low background upon transformation of the ligation product intobacteria.

Two single-stranded oligonucleotide sequences (see below) aresynthesized, annealed, and repetitively filled in, denatured, andreannealed to form double-stranded oligonucleotides that, when digestedwith AflII and BclI, can be ligated into the polylinker of theexpression vector, Cadus 1215. The two single-stranded oligonucleotideshave the following sequences:

5′-G CTA CTT AAG CGT GAG GCA GAA GCT 3′ and (SEQ ID NO:13) 5′-C GGA TGATCA (NNN)_(n)AGC TTC TGC CTC ACG CTT AAG TAG C 3′ (SEQ ID NO:14)where N is any chosen nucleotide and n is any chosen integer. Yeasttransformed with the resulting plasmids will secrete—through theα-factor secretory pathway—peptides whose amino acid sequence isdetermined by the particular choice of N and n).Alternatively, the following single stranded oligonucleotides are used:MFαNNK (76 mer):5′CTGGATGCGAAGACAGCTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNK NNKTGATCAGTCTGTGACGC 3′ (SEQ ID NO:15)and MFαMbo (17 mer):5′ GCGTCACAGACTGATCA 3′ (SEQ ID NO:16)When annealed the double stranded region is:

TGATCAGTCTGTGACGC (SEQ ID NO:17) ACTAGTCAGACACTGCG (SEQ ID NO:18)

After fill-in using Taq DNA polymerase (Promega Corporation, Madison,Wis.), the double stranded product is restricted with BbsI and MboI andligated to HindIII- and BglII-restricted Cadus 1373.

ii. The region of MFα1 which encodes mature a-factor will be replacedvia single stranded mutagenesis with restriction sites that can acceptoligonucleotides with XhoI and AflII ends. Insertion of oligonucleotideswith XhoI and AflII ends will yield plasmids which encode proteinscontaining the MFα1 leader sequences upstream of the sequence encoded bythe oligonucleotides. The MFα1 leader sequences should direct theprocessing of these precursor proteins through the pathway normally usedfor the transport of mature a-factor. MFA1, obtained as a BamHI fragmentfrom pKK1 (provided by J. 30 Thorner and K. Kuchler), was ligated intothe BamHI site of pALTER (Promega). Using the minus strand of MFA1 astemplate, a HindIII site was inserted by oligonucleotide-directedmutagenesis just 5′ to the MFA1 start codon using the followingoligonucleotide:

5′CCAAAATAAGTACAAAGCTTTCGAATAGAAATGCAACCATC (SEQ ID NO:19)

A second oligonucleotide was used simultaneously to introduce a shortpolylinker for later cloning of synthetic oligonucleotides in place ofMFA1 sequences. These MFA1 sequences encode the C-terminal 5 amino acidsof the 21 amino acid leader peptide through to the stop codon:

5′GCCGCTCCAAAAGAAAAGACCTCGAGCTCGCTTAAGTTCTGCGTACAAAAACG-TTGTTC 3′ (SEQID NO:20)

The 1.6 kb HindIII fragment of the resulting plasmid, Cadus 1172,contains sequences encoding the MFA1 start codon and the N-terminal 16amino acids of the leader peptide, followed by a short polylinkercontaining XhoI, SacI, and AflII sites for insertion ofoligonucleotides. The 1.6 kb HindIII fragment of Cadus 1172 was ligatedinto HindIII-digested Cadus 1186 (see above) to place expression ofsequences cloned into this cassette under the control of the ADH1promoter. The SacI site in the polylinker was made unique by eliminatinga second SacI site present in the vector. The resulting plasmid,designated Cadus 1239, can be prepared to accept oligonucleotides withXhoI and AflII ends by digestion with those restriction endonucleasesfor expression in the context of MFα1 leader peptides (FIG. 3).

Two single-stranded oligonucleotide sequences (see below) aresynthesized, annealed, and repetitively filled in, denatured, andreannealed to form double-stranded oligonucleotides that, when digestedwith AflII and BglII, can be cloned into the polylinker of theexpression vector, Cadus 1239. The two single-stranded oligonucleotidesused for the cloning have the following sequences:

5′GG TAC TCG AGT GAA AAG AAG GAC AAC 3′ (SEQ ID NO:21) 5′CG TAC TTA AGCAAT AAC ACA (NNN)_(n)GTT GTC CTT CTT TTC ACT CGA GTA CC 3′ (SEQ IDNO:22)where N is any chosen nucleotide and n is any chosen integer.Yeast transformed with the resulting plasmids will transport—through thepathway normally used for the export of a-factor—farnesylated,carboxymethylated peptides whose amino acid sequence is determined bythe particular choice of N and n (FIG. 3).

Example 4 Peptide Secretion/Transport

This example demonstrates the ability to engineer yeast such that theysecrete or transport oligonucleotide-encoded peptides (in this casetheir pheromones) through the pathways normally used for the secretionor transport of endogenous pheromones.

Autocrine MATa Strain CY588:

A MATa strain designed for the expression of peptides in the context ofMFα1 (i.e., using the MFα1 expression vector, Cadus 1215) has beenconstructed. The genotype of this strain, which we designate CY588, isMATa bar1 far1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3. The barlmutation eliminates the strain's ability to produce a protease thatdegrades α-factor and that may degrade some peptides encoded by thecloned oligonucleotides; the far1 mutation abrogates the arrest ofgrowth which normally follows stimulation of the pheromone responsepathway; an integrated FUS1-HIS3 hybrid gene provides a selectablesignal of activation of the pheromone response pathway; and, finally,the stel4 mutation lowers background of the FUS1-HIS3 readout. Theenzymes responsible for processing of the MFa1 precursor in MATα cellsare also expressed in MATa cells (Sprague and Thomer, in The Molecularand Cellular Biology of the Yeast Saccharomyces: Gene Expression, 1992,Cold Spring Harbor Press), therefore, CY588 cells should be able tosecrete peptides encoded by oligonucleotides expressed from plasmidCadus 1215.

A high transforming version (tbtl-1) of CY588 was obtained by crossingCY1013 (CY588 containing an episomal copy of the STE14 gene) (MATabarl::hisGfar1-1fusl-HIS3 stel4::TRP1 ura3 trpl leu2 his3 [STE14 URA3CEN4) to CY793 (MATα˜tbtl-1 ura3 leu2 trpl his3 fus1-HIS2 can1ste114::TRP1 [FUS1 LEU2μ]) and selecting from the resultant spores astrain possessing the same salient genotype described for CY588 (seeabove), and in addition the tbl-1 allele, which confers the capacity forvery high efficiency transformation by electroporation. The selectedstrain is CY1455 (MATabarl::hisGfar1-1 fus1-HIS3 ste14::TRP1 tbt-1 ura3trpl leu2 his3).

Secretion of peptides in the context of yeast α-factor:

Experiments were performed to test: 1. the ability of Cadus 1215 tofunction as a vector for the expression of peptides encoded by syntheticoligonucleotides; 2. the suitability of the oligonucleotides, asdesigned, to direct the secretion of peptides through the α-factorsecretory pathway; 3. the capacity of CY588 to secrete those peptides;and 4. the ability of CY588 to respond to those peptides that stimulatethe pheromone response pathway by growing on selective media. Theseexperiments were performed using an oligonucleotide which encodes the 13amino acid α-factor; i.e., the degenerate sequence (NNN)_(n) in theoligonucleotide cloned into Cadus 1215 (see above) was specified (n=13)to encode this pheromone. CY588 was transformed with the resultingplasmid (Cadus 1219), and transformants selected on uracil-deficientmedium were transferred to histidine-deficient medium supplemented witha range of concentrations of aminotriazole (an inhibitor of the HIS3gene product that serves to reduce background growth). The resultsdemonstrate that the synthetic oligo-nucleotide, expressed in thecontext of MFα1 by Cadus 1215, conferred upon CY588 an ability to growon histidine-deficient media supplemented with aminotriazole. Insummation, these data indicate that: 1. CY588 is competent for thesecretion of a peptide encoded by the (NNN)_(n) sequence of thesynthetic oligonucleotide cloned into and expressed from Cadus 1215; and2. CY588 can, in an autocrine fashion, respond to a secreted peptidewhich stimulates its pheromone response pathway, in this case byα-factor binding to STE2.

Additional experiments were performed to test the utility of autocrineyeast strains in identifying agonists of the Ste2 receptor from amongmembers of two semi-random α-factor libraries, α-Mid-5 and MFα-8.

α-Mid-5 Library

A library of semi-random peptides, termed the α-Mid-5 library, wasconstructed. In this library, the N-terminal four amino acids and theC-terminal four amino acids of a 13 -residue peptide are identical tothose of native α-factor while the central five residues (residues 5–9)are encoded by the degenerate sequence (NNQ)₅. The followingoligonucleotides were used in the construction of the α-Mid-5 library:

(1) MFaMbo, a 17 mer:

5′ GCGTCACAGACTGATCA (SEQ ID NO:23)

(2) MID5aLF, a 71 mer:

5′

GCCGTCAGTAAAGCTTGGCATTGGTTGNNQNNQNNQNNQMMQCAGCCTATGTA CTGATCAGTCTGTGACGC (SEQ ID NO:24)

Sequenase (United States Biochemical Corporation, Cleveland, Ohio) wasused to complete the duplex formed after annealing MFaMbo to the MID5aLFoligonucleotide. In the MID5aLF sequence, N indicates a mixture of A, C,G, and T at ratios of 0.8:1:1.3:1; Q indicates a mixture of C and G at aratio of 1:1.3. These ratios were employed to compensate for thedifferent coupling efficiences of the bases during oligonucleotidesynthesis and were thus intended to normalize the appearance of allbases in the library. The double-stranded oligonucleotide was restrictedwith HindIII and MboI and ligated to Cadus 1625 (see above); Cadus 1625had been prepared to accept the semi-random oligonucleotides byrestriction with HindIII and BglII.

The apparent complexity of the αMid-5 library is 1×10⁷. This complexityis based on the number of bacterial transformants obtained with thelibrary DNA versus transformants obtained with control vector DNA thatlacks insert. Sequence analysis of six clones from the librarydemonstrated that each contained a unique insert.

To identify peptide members of the α-mid-5 library that could act asagonists on the STE2 receptor, CY1455, a high transforming version ofCY588, was electroporated to enhance uptake of α-Mid-5 DNA.Transformants were selected on uracil-deficient (-Ura) syntheticcomplete medium and were transferred to histidine-deficient (-His)synthetic complete medium supplemented with 0.5 mM or 1 mMaminotriazole.

Yeast able to grow on -His+ aminotriazole medium include (1) yeast whichare dependent on the expression of an α-factor variant agonist and (2)yeast which contain mutations that result in constitutive signallingalong the pheromone pathway. Yeast expressing and secreting a variantSTE2 receptor agonist have the ability to stimulate the growth on -Hismedium of surrounding CY 1455 cells that do not express such an agonist.Thus a recognizable formation (termed a “starburst”) results, consistingof a central colony, growing by virtue of autocrine stimuation of thepheromone pathway, surrounded by satellite colonies, growing by virtueof paracrine stimulation of the pheromone pathway by the agonist peptideas that peptide diffuses radially from the central, secreting colony.

In order to identify the peptide sequence responsible for this“starburst” phenomenon, yeast were transferred from the center of the“starburst” and streaks were made on -Ura medium to obtain singlecolonies. Individual clones from -Ura were tested for the His+ phenotypeon -His+ aminotriazole plates containing a sparse lawn of CY1455 cells.Autocrine yeast expressing a peptide agonist exhibited the “starburst”phenotype as the secreted agonist stimulated the growth of surroundingcells that lacked the peptide but were capable of responding to it.Constitutive pheromone pathway mutants were capable of growth on -His+aminotriazole but were incapable of enabling the growth of surroundinglawn cells.

Alternatively, streaks of candidate autocrine yeast clones were made onplates containing 5-fluoroorotic acid (FOA) to obtain Ura segregantswere retested on -His+ aminotriazole for the loss of the His+ phenotype.Clones that lost the ability to grow on -His+ aminotriazole afterselection on FOA (and loss of the peptide-encoding plasmid) derived fromcandidate expressors of a peptide agonist. The plasmid was rescued fromcandidate clones and the peptide sequences determined. In addition, aplasmid encoding a putative Ste2 agonist was reintroduced into CY1455 toconfirm that the presence of the plasmid encoding the peptide agonistconferred the His+ phenotype to CY1455.

By following the above protocol novel Ste2 agonists have been identifiedfrom the α-Mid-5 library. Sequences of nine agonists follow, preceded bythe sequence fo the native α-factor pheromone and by the oligonucleotideused to encode the native pheromone in these experiments. (Note thevariant codons used in the α-factor-encoding oligonucleotide forglutamine and proline in the C-terminal amino acids of α-factor).

α-factor TGG CAT TGG TTG CAG CTA AAA CCT GGC CAA CCA ATG TAC (SEQ IDNO:25) encodes  Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr (SEQID NO:26) α-factor oligo:  TGG CAT TGG TTG CAG CTA AAA CCT GGC CAG CCTATG TAC (SEQ ID NO:27) encodes Trp His Trp Leu Gln Leu Lys Pro Gly GlnPro Met Tyr (SEQ ID NO:28) M1      TGG CAT TGG TTG TCC TTG TCG CCC GGGCAG CCT ATG TAC (SEQ ID NO:29) encodes Trp His Trp Leu Ser Leu Ser ProGly Gln Pro Met Tyr (SEQ ID NO:30) M2      TGG CAT TGG TTG TCC CTG GACGCT GGC CAG CCT ATG TAC (SEQ ID NO:31) encodes Trp His Trp Leu Ser LeuAsp Ala Gly Gln Pro Met Tyr (SEQ ID NO:32) M3      TGG CAT TGG TTG ACCTTG ATG GCC GGG CAG CCT ATG TAC (SEQ ID NO:33) encodes Trp His Trp LeuThr Leu Met Ala Gly Gln Pro Met Tyr (SEQ ID NO:34) M4      TGG CAT TGGTTG CAG CTG TCG GCG GGC CAG CCT ATG TAC (SEQ ID NO:35) encodes Trp HisTrp Leu Gln Leu Ser Ala Gly Gln Pro Met Tyr (SEQ ID NO:36) M5      TGGCAT TGG TTG AGG TTG CAG TCC GGC CAG CCT ATG TAC (SEQ ID NO:37) encodesTrp His Trp Leu Arg Leu Gln Ser Gly Gln Pro Met Tyr (SEQ ID NO:38)M6      TGG CAT TGG TTG CGC TTG TCC GCC GGG CAG CCT ATG TAC (SEQ IDNO:39) encodes Trp His Trp Leu Arg Leu Gln Ser Gly Gln Pro Met Tyr (SEQID NO:40) M7      TGG CAT TGG TTG TCG CTC GTC CCG GGG CAG CCT ATG TAC(SEQ ID NO:41) encodes Trp His Trp Leu Ser Leu Val Pro Gly Gln Pro MetTyr (SEQ ID NO:42) M8      TGG CAT TGG TTG TCC CTG TAC CCC GGG CAG CCTATG TAC (SEQ ID NO:43) encodes Trp His Trp Leu Ser Leu Tyr Pro Gly GlnPro Met Tyr (SEQ ID NO:44) M9      TGG CAT TGG TTG CGG CTG CAG CCC GGGCAG CCT ATG TAC (SEQ ID NO:45) encodes Trp His Trp Leu Arg Leu Gln ProGly Gln Pro Met Tyr (SEQ ID NO:46)

The nine peptide agonists of the Ste2 receptor above were derived fromone electroporation of CY1455 using 1 μg of the α-Mid-5 library DNA.Approximately 3×10⁵ transformants were obtained, representingapproximately 0.03% of the sequences present in that library.

MFα-8 Library

A semi-random α-factor library was obtained through synthesis ofmutagenized α-factor oligonucleotides such that 1 in 10,000 peptideproducts were expected to be genuine α-factor. The mutagenesis wasaccomplished with doped synthesis of the oligonucleotides: eachnucleotide was made approximately 68% accurate by synthesizing thefollowing two oligos:

5′CTGGATGCGAAGACTCAGCT (SEQ ID NO:47) (20 mer) (oligo060)

5′CGGATGATCA gta cat tgg ttg gcc agg ttt tag ctg caa cca atg cca AGC TGAGTC TTC GCA TCC AG (69 mer) (oligo074) (SEQ ID NO:48)

The lower case letters indicate a mixture of 67% of that nucleotide and11% of each of the other three nucleotides (e.g. t indicates 67% T and11% A, 11% C, and 11% G). Note that digestion of the double-strandedoligo-nucleotide by Foki or BbsI will yield an identical 5′ end that iscompatible with HindIII ends.Oligos 060 and 074 will form the following double-stranded molecule whenannealed:

5′-CTGGATGCGAAGACTCAGCTc (SEQ ID NO:49) 3′-GACCTACGCTTCTGAGTCGA acc gtaacc aac gtc gat ttt gga ccg gtt ggt tac atg ACTAGTAGGC-5′ (SEQ ID NO:50)

The duplex was repetitively filled-in using Taq DNA polymerase (PromegaCorporation, Madison, Wis.). The double-stranded product was restrictedwith BbsI and BclI and ligated into HindIII- and BglII-digested Cadus1373. The BglII/BclI joint creates a TGA stop codon for the terminationof translation of the randomers. Using this approach, the MFα-5.8library (a library of apparent low complexity based on PCR analysis ofoligonucleotide insert frequency) was constructed.

To identify peptide members of the MFα-5.8 library that could act asagonists on the STE2 receptor, CY1455, a high transforming version ofCY588, was electroporated to enhance uptake of MFα-5.8 DNA.Transformants were selected on uracil-deficient (-Ura) syntheticcomplete medium and were transferred to histidine-deficient (-His)synthetic complete medium supplemented with 1.0 mM or 2.5 mMaminotriazole. Yeast from colonies which were surrounded by satellitegrowth were transferred as streaks to -Ura medium to obtain singlecolonies. Yeast from single colonies wree then tested for the His+phenotype on -His+ aminotriazole plates. Sequence analysis of seven ofthe plasmids rescued from His+ yeast revealed three unique α-factorvariants that acted as agonists on the STE2 receptor.

1.4 independent clones had the following sequence:

(SEQ ID NO:51) TGG CAT TGG CTA CAG CTA ACG CCT GGG CAA CCA ATG TACencoding (SEQ ID NO:52) Trp His Trp Leu Gln Leu Thr Pro Gly Gln Pro MetTyr2.2 independent clones had the following sequence:

(SEQ ID NO:53) TGG CAT TGG CTG GAG CTT ATG CCT GGC CAA CCA TTA TACencoding (SEQ ID NO:54) Trp His Trp Leu Glu Leu Met Pro Gly Gln Pro LeuTyr

3. (SEQ ID NO:55) TGG CAT TGG ATG GAG CTA AGA CCT GGC CAA CCA ATG TACencoding (SEQ ID NO:56) Trp His Trp Met Glu Leu Arg Pro Gly Gln Pro MetTyrAutocrine Mata strain CY599:

A MATA strain designed for the expression of peptides in the context ofMFA1 (i.e., using the MFA1 expression vector, Cadus 1239) has beenconstructed. The genotype of this strain, designated CY599, is MATa mfalmfa2 far1-1 his3::fus1-HIS3 ste2-STE3 ura3 metl adel leu2. In thisstrain, Cadus 1147 (see above) was used to replace STE2 with a hybridgene in which the STE3 coding region is under the control of expressionelements which normally drive the expression of STE2. As a result, thea-factor receptor replaces the α-factor receptor. The genes which encodea-factor are deleted from this strain; the far1 mutation abrogates thearrest of growth which normally follows stimulation of the pheromoneresponse pathway; and the FUS1-HIS3 hybrid gene (integrated at the HIS3locus) provides a selectable signal of activation of the pheromoneresponse pathway. CY599 cells were expected to be capable of thetransport of a-factor or a-factor-like peptides encoded byoligonucleotides expressed from Cadus 1239 by virtue of expression ofthe endogenous yeast transporter, Ste6.

Transport of Peptides by the Yeast a-Factor Pathway:

Experiments were performed to test: 1. the ability of Cadus 1239 tofunction as a vector for the expression of peptides encoded by syntheticoligonucleotides; 2. the suitability of the oligonucleotides, asdesigned, to direct the export of farnesylated, carboxymethylatedpeptides through the pathway normally used by a-factor; 3. the capacityof CY599 to export these peptides; and 4. the ability of CY599 torespond to those peptides that stimulate the pheromone response pathwayby growing on selective media. These tests were performed using anoligonucleotide which encodes the 12 amino acid a-factor; specifically,the degenerate sequence (NNN)_(n) in the oligo-nucleotide cloned intoCadus 1239 (see above) (with n=12) encodes the peptide component ofa-factor pheromone. CY599 was transformed with the resulting plasmid(Cadus 1220), and transformants selected on uracil-deficient medium weretransferred to histidine-deficient medium supplemented with a range ofconcentrations of aminotriazole. The results demonstrate that thesynthetic oligonucleotide, expressed in the context of MFA1 by Cadus1220, conferred upon CY599 enhanced aminotriazole-resistant growth onhistidine-deficient media. In summation, these data indicate: 1. Cadus1220 and the designed oligonucleotide are competent to direct theexpression and export of a farnesylated, carboxymethylated peptideencoded by the (NNN)_(n) sequence of the synthetic oligonucleotide; and2. CY599 can, in an autocrine fashion, respond to a farnesylated,carboxymethylated peptide that stimulates its pheromone responsepathway, in this case signaling initiates as a-factor binds to STE3.

Example 5 Proof of Concept

This example will demonstrate the utility of the autocrine system forthe discovery of peptides which behave as functional pheromoneanalogues. By analogy, this system can be used to discover peptides thatproductively interact with any pheromone receptor surrogates.

CY588 (see strain 5, Example 2 above) will be transformed with CADUS1215 containing oligonucleotides encoding random tridecapeptides for theisolation of functional a factor analogues. CYS99 (see strain 6, Example2 above) will be transformed with CADUS 1239 containing oligos of randomsequence for the isolation of functional a-factor analogues. Colonies ofeither strain which can grow on histidine-deficient media followingtransformation will be expanded for the preparation of plasmid DNA, andthe oligo-nucleotide cloned into the expression plasmid will besequenced to determine the amino acid sequence of the peptide whichpresumably activates the pheromone receptor. This plasmid will then betransfected into an isogenic strain to confirm its ability to encode apeptide which activates the pheromone receptor. Successful completion ofthese experiments will demonstrate the potential of the system for thediscovery of peptides which can activate membrane receptors coupled tothe pheromone response pathway.

Random oligonucleotides to be expressed by the expression plasmid CADUS1215 will encode tridecapeptides constructed as

5′CGTGAAGCTTAAGCGTGAGGCAGAAGCT(NNK)₁₃TGATCATCCG (SEQ ID NO:57), where Nis any nucleotide, K is either T or G at a ratio of 40:60 (see Proc.Natl. Acad. Sci. 87:6378, 1990; ibid 89:5393, 1992), and the AflII andBclI sites are underlined. This oligonucleotide is designed such that:the AflII and BclI sites permit inserting the oligos into the AflII andBglII site of CADUS 1215, the HindIII site just 5′ to the AflII site inthe 5′ end of the oligo allows future flexibility with cloning of theoligos; the virtual repeat of GAGGCT and the GAGA repeats which arepresent in the wild-type sequence and which can form triple helixes arechanged without altering the encoded amino acids. The randomoligonucleotides described above will actually be constructed from thefollowing two oligos:

5′ CGTGAAGCTTAAGCGTGAGGCAGAAGCT (SEQ ID NO:58) and 5′CGGATGATCA(MNN)₁₃AGCTTCTG (SEQ ID NO:59),where M is either A or C at a ratio of 40:60. The oligos will beannealed with one another and repetitively filled in, denatured, andreannealed (Kay et al, Gene, 1993). The double-stranded product will becut with AflII and BclI and ligated into the AflII- and BglII-digestedCADUS 1215. The BglII/BclI joint will create a TGA stop codon fortermination of translation of the randomers. Because of the TA contentof the Afl overhang, the oligos will be ligated to the AflII-andBglII-digested pADC-MFα at 4° C.

Random oligonucleotides to be expressed by the expression plasmid CADUS1239 will encode monodecapeptides constructed as

5′ GGTACTCGAGTGAAAAGAAGGACAAC(NNK)₁₁TGTGTTATTGCTTAAGTACG (SEQ ID NO:60),where N is any nucleotide, K is either T or G at a ratio of 40:60 (seeProc. Natl. Acad. set 87:6378, 1990; ibid 89:5393, 1992). When clonedinto the XhoI and AflII sites of CADUS 1239 the propeptides expressedunder the control of the ADH1 promoter will contain the entire leaderpeptide of MFal, followed by 11 random amino acids, followed by tripletsencoding CVIA (the C-terminal tetrapeptide of wild-type a-factor).Processing of the propeptide should result in the secretion ofdodecapeptides which contain 11 random amino acids followed by aC-terminal, farnesylated, carboxymethylated cysteine.

Using the procedure described above, the oligonucleotides for expressionin CADUS 1239 will actually be constructed from the following twooligos:

5′ GGTACTCGAGTGAAAAGAAGGACAAC (SEQ ID NO:61) and 5′ (SEQ ID NO:62),CGTACTTAAGCAATAACAca(MNN)₁₁GTTGTCC

where M is either A or C at a ratio of 40:60, and the XhoI and AflIIsites are underlined.

Discovery of a-Factor Analoques from a Random Peptide Library

An optimized version of strain 6 (Example 2 above) was derived. Thisyeast strain, CY2012 (MATa ste2-STE3 far1Δl442 mfal::LEU2 mfa2-lacZfusl-HIS3 tbtl-1 ura3 leu2 his3 trpl suc2), was constructed as follows.From a cross of CY570 (MATa mfal::LEU2 mfa2-lacZ ura3 trpl his3Δ200 can1leu2 fus1-HIS3 [MFA] URA3 2μ] [Fus1Δ8–73 TRP1 CEN6]) by CY1624(MATαtbtl-1 fus1-HIS3 trpl ura3 leu2 his3 lys2-801 SUC+), a spore wasselected (CY1877) of the following genotype: MATa mfal::LEU2 mfa2-lacZfus1-HIS3 tbtl-1 ura3 leu2 his3 trp1 suc2. This strain lacks both genes(NFA1 and MFA2) encoding a-factor precursors, contains the appropriatepheromone pathway reporter gene (fusl-HIS3), and transforms byelectroporation at high efficiency (tbtl-1). This strain was altered bydeletion of the FAR1 gene (with Cadus 1442; see Example 6), andreplacement of STE2 coding sequences with that of STE3 (see Example 1)to yield CY2012.

This strain was transformed with plasmid DNA from a random α-factorlibrary by electroporation and plated on 17 synthetic complete plateslacking uracil (-Ura), yielding approximately 10⁵ Ura+colonies per plateafter 2 days at 30° C. These colonies were replica plated tohistidine-deficient synthetic complete media (-His) containing 0.2 mM3-aminotriazole and after three days at 30° C. 35 His+ replicas werestreaked to -Ura plates. The resultant colonies, 3 from each isolate,were retested for their His+ phenotype, and streaked to 5-fluorooroticacid plates to obtain Ura segregants (lacking a library plasmid). ThoseUra-segregants were tested for the loss of their His+ phenotype. Ten ofthe original isolates passed these tests; in two cases only one of thethree Ura+ colonies purified from the isolate retained the His+phenotype, but nevertheless subsequently segregated Ura His- colonies.

A single plasmid (corresponding to a bacterial colony) was obtained fromeach of the ten isolates, and reintroduced into CY2012. Eight of the tenplasmids passed the test of retaining the ability to confer the His+phenotype on CY2012 (the two that failed correspond to the two isolatesthat were mentioned above, suggesting that these isolates contain atleast one “irrelevant” plasmid). Sequencing of the randomized insert inthe eight plasmids of interest revealed that four contain the sequence:

(SEQ ID NO:63) TAT GCT CTG TTT GTT CAT TTT TTT GAT ATT CCG (SEQ IDNO:64) Tyr Ala Leu Phe Val His Phe Phe Asp Ile Pro

two contain the sequence:

(SEQ ID NO:65) TTT AAG GGT CAG GTG CGT TTT GTG GTT CTT GCT (SEQ IDNO:66) Phe Lys Gly Gln Val Arg Phe Val Val Leu Ala,

and two contain the sequence:

(SEQ ID NO:67) CTT ATG TCT CCG TCT TTT TTT TTT TTG CCT GCG (SEQ IDNO:68) Leu Met Ser Pro Ser Phe Phe Phe Leu Pro AlaClearly, these sequences encode novel peptides, as the native α-factorsequence differs considerably:

Tyr Ile Ile Lys Gly Val Phe Trp Asp Pro Ala. (SEQ ID NO:69)

The a-factor variants identified from random peptide libraries haveutility as “improved” substrates of ABC transporters expressed in yeast.For example, identification of a preferred substrate of human MDR, onethat retains agonist activity on the pheromone receptor, would permitthe establishment of robust yeast screens to be used in the discovery ofcompounds that affect transporter function.

Example 6 Functional Expression of a Mammalian G Protein-CoupledReceptor and Ligand in an Autocrine Yeast Strain

This example details the following: (1) expression of human C5a receptorin yeast; (2) expression of the native ligand of this receptor, humanC5a, in yeast; and (3) activation of the endogenous yeast pheromonepathway upon stimulation of the C5a receptor by C5a when both of thesemolecules are expressed within the same strain of autocrine yeast.Following the experimental data we outline the utility of autocrinestrains of yeast that functionally express the human C5a receptor.

Human C5a is a 74 amino acid polypeptide that derives from the fifthcomponent of complement during activation of the complement cascade; itis the most potent of the complement-derived anaphylatoxins. C5a is apowerful activator of neutrophils and macrophage functions includingproduction of cytotoxic super oxide radicals and induction of chemotaxisand adhesiveness. In addition C5a stimulates smooth muscle contraction,induces degranulation of mast cells, induces serotonin release fromplatelets and increases vascular permeability. The C5a anaphylatoxin canalso amplify the inflammatory response by stimulating the production ofcytokines. As C5a is a highly potent inflammatory agent, it is a primarytarget for the development of antagonists to be used for intervention ina variety of inflammatory processes.

The C5a receptor is present on neutrophils, mast cells, macrophages andsmooth muscle cells and couples through G proteins to transmit signalsinitiated through the binding of C5a.

Expression of the C5a Receptor

The plasmid pCDM8-C5aRc, bearing cDNA sequence encoding the human C5areceptor, was obtained from N. Gerard and C. Gerard (Harvard MedicalSchool, Boston, Mass.) (Gerard and Gerard 1991). Sequence encoding C5awas derived from this plasmid by PCR using VENT polymerase (New EnglandBiolabs Inc., Beverly Mass.), and the following primers:

#1-GGTGGGAGGGTGCTC T CTAGAAGGAAGTGTTCACC (SEQ ID NO:70)#2-GCCCAGGAGACCAGA C C ATGG ACTCCTTCAATTATACCACC (SEQ ID NO:71)Primer #1 contains a single base-pair mismatch (underlined) to C5areceptor cDNA. It introduces an XbaI site (in bold) 201 bp downstreamfrom the TAG termination codon of the C5a receptor coding sequence.Primer #2 contains two mismatched bases and serves to create an NcoIsite (in bold) surrounding the ATG initiator codon (double underlined).The second amino acid is changed from an aspartic acid to an asparagineresidue. This is the only change in primary amino acid sequence from thewild type human C5a receptor.

The PCR product was restricted with NcoI and XbaI (sites in bold) andcloned into CADUS 1002 (YEp51Nco), a Gal10 promoter expression vector.The sequence of the entire insert was determined by dideoxy sequencingusing multiple primers. The sequence between the NcoI and XbaI sites wasfound to be identical to the human C5a receptor sequence that wasdeposited in GenBank (accession #JO5327) with the exception of thosechanges encoded by the PCR primers. The C5a receptor-encoding insert wastransferred to CADUS 1289 (pLPXt), a PGK promoter expression vector,using the NcoI and XbaI sites, to generate the C5a receptor yeastexpression clone, CADUS 1303.

A version of the C5a receptor which contains a yeast invertase signalsequence and a myc epitope tag at its amino terminus was expressed inCadus 1270-transferred yeast under control of a GAL10 promoter. Plasmidsencoding an untagged version of the C5a receptor and a myc-taggedderivative of FUS1 served as controls. The expression of the taggedreceptor in yeast was confirmed by Western blot using the anti-mycmonoclonal antibody 9E10. In the lane containing the extract from theCadus 1270-transformant, the protein that is reactive with the anti-mycmonoclonal antibody 9E10 was approximately 40 kD in size, as expected.Note that this receptor construct is not identical to the one used inthe autocrine activation experiments. That receptor is not tagged, doesnot contain a signal sequence and is driven by the PGK promoter.

Expression of the Ligand, C5a

A synthetic construct of the sequence encoding C5a was obtained from C.Gerard (Harvard Medical School, Boston, Mass.). This synthetic gene hadbeen designed as a FLAG-tagged molecule for the secretion from E. coli(Gerard and Gerard (1990) Biochemistry 29:9274–9281). The C5a codingregion, still containing E. coli codon bias, was amplified using VENTpolymerase (New England Biolabs Inc., Beverly Mass.) through 30 cyclesusing the following primers:

C5a5′ = CCCCTTAAGCGTGAGGCAGAAGCTACTCTGCAAAAGAAGATC (SEQ ID NO:72) C5a3′= GAAGATCTTCAGCGGCCGAGTTGCATGTC (SEQ ID NO:73)

A PCR product of 257 bp was gel isolated, restricted with AflII andBglII, and cloned into CADUS 1215 (an expression vector designed toexpress peptide sequences in the context of Mfα) to yield CADUS 1297.The regions of homology to the synthetic C5a gene are underlined. The 5′primer also contains pre-pro α-factor sequence. Upon translation andprocessing of the pre-pro α-factor sequence, authentic human C5a shouldbe secreted by yeast containing CADUS 1297. The insert sequence in CADUS1297 was sequenced in both orientations by the dideoxy method and foundto be identical to that predicted by the PCR primers and the publishedsequence of the synthetic C5a gene (Franke et al. (1988) Methods inEnzymology 162: 653–668).

Two sets of experiments, aside from the autocrine activation of yeastdetailed below, demonstrated that CADUS 1297 can be used to express C5ain yeast.

1). C5a was immunologically detected in both culture supernatant andlysed cells using a commercially available enzyme-linked immunosorbentassay (ELISA)(Table 1). This assay indicated the concentration of C5a inthe culture supernatant to be approximately 50 to 100 nM. In comparison,in data derived from mammalian cells, the binding constant of C5a to itsreceptor is 1 nM (Boulay et al.(1991) Biochemistry 30:2993–2999.

2). C5a expressed in yeast was shown to compete for binding withcommercially obtained (Amersham Corporation, Arlington Heights, Ill.),radiolabeled C5a on induced HL60 cells.

Activation of the Pheromone Response Pathway in Autocrine YeastExpressing the Human C5a Receptor and Human C5a

Activation of the yeast pheromone response pathway through theinteraction of C5a with the C5a receptor was demonstrated using a growthread-out. The strain used for this analysis, CY455 (MATα tbt1-1 ura3leu2 trp1 his3 fusl-HIS3 can1 ste14::TRP1 ste3*1156) contains thefollowing significant modifications. A pheromone inducible HIS3 gene,fus1-HIS3, is integrated at the Fus1 locus. A hybrid gene containingsequence encoding the first 41 amino acids of GPA1 (the yeast Gαsubunit) fused to sequence encoding human Gαi2A (minus codons for theN-terminal 33 amino acids) replaces GPA1 at its normal chromosomallocation. The yeast STE14 gene is disrupted to lower the basal level ofsignaling through the pheromone response pathway. The yeast α-factorreceptor gene, STE3, is deleted. The last two modifications are probablynot essential, but appear to improve the signal-to-noise ratio.

CY455 (MATα tbt1-1 ura3 leu2 trpl his3 fusl-HIS3 can1 stel4::TRP1ste3*1156) was transformed with the following plasmids:

Cadus 1289+Cadus 1215=Receptor⁻ Ligand⁻=(R−L−)

Cadus 1303+Cadus 1215=Receptor⁺ Ligand⁻=R+L−

Cadus 1289+Cadus 1297=Receptor⁻ Ligand⁺=(R−L+).

Cadus 1303+Cadus 1297=Receptor⁺ Ligand⁺=(R+L+)

Receptor refers to the human C5a receptor.

Ligand refers to human C5a.

Three colonies were picked from each transformation and grown overnightin media lacking leucine and uracil, at pH 6.8 with 25 mM PIPES (LEU URApH6.8 with 25 mM PIPES). This media was made by adding 0.45 ml ofsterile 1M KOH and 2.5 ml of sterile 1M PIPES pH 6.8 to 100 ml ofstandard SD LEU- URA- media. After overnight growth the pH of this mediais usually acidified to approximately pH 5.5. Overnight cultures werewashed once with 25 mM PIPES pH 6.8 and resuspended in an equal volumeof media lacking leucine, uracil and histidine (LEU URA HIS pH 6.8 with25 mM PIPES). The optical density at 600 nm of a 1/20 dilution of thesecultures was determined and the cultures were diluted into 25 mM PIPESpH 6.8 to a final OD₆₀₀ of 0.2. A volume (5 ul) of this dilutionequivalent to 10,000 cells was spotted onto selective (HIS+ TRP− pH6.8)plates. Only those strains expressing both C5a and its receptor (R+L+)show growth on the selective plates which lack histidine. All teststrains are capable of growth on plates containing histidine. TheR+L+strain will grow on plates containing up to 5 mM aminotriazole, thehighest concentration tested.

For verification of pheromone pathway activation and quantification ofthe stimulation, the activity of the fusl promoter was determinedcolorometrically using a fusl-lacZ fusion in a similar set of strains.CY878 (MATα tbtl-1 fusl-HIS3 caNI stel4::Trpl::LYS2 ste3*1156gpal(41)-Gαi2) was used as the starting strain for these experiments.This strain is a trpl derivative of CY455. The transformants for thisexperiment contained CADUS 1584 (pRS424-fusl-lacZ) in addition to thereceptor and ligand plasmids. Four strains were grown overnight in SDLEU URA TRP pH6.8 with 50 mM PIPES to an OD₆₀₀ of less than 0.8. Assayof β-galactosidase activity (Guarente 1983) in these strains yields thedata shown in FIG. 4. The coupling of the C5a receptor to Gα chimeras isshown in Table 2.

Projected Uses of the Autocrine C5a Strains:

A primary use of the autocrine C5a strains will be in the discovery ofC5a antagonists. Inhibitors of the biological function of C5a would beexpected to protect against tissue damage resulting from inflammation ina wide variety of inflammatory disease processes including but notlimited to: respiratory distress syndrome (Duchateau et al. (1984) AmRev Respir Dis 130:1058); (Hammerschmidt et al. (1980) Lancet 1:947),septic lung injury (Olson et al. 1985) Ann Surg 202:771), arthritis(Banerjee et al. (1989) J. Immuinol 142:2237), ischemic andpost-ischemic myocardial injury (Weisman (1990) Science 146:249);(Crawford et al. (1988) Circulation 78:1449) and burn injury (Gelfand etal. (1982) J. Clin Invest 70:1170).

The autocrine C5a system as described can be used to isolate C5aantagonists as follows:

1. High throughput Screens to Identify Antagonists of C5a.

A straightforward approach involves screening compounds to identifythose which inhibit growth of the R+L+ strain described above inselective media but which do not inhibit the growth of the same strainor of a R+L− strain in non-selective media. The counterscreen isnecessary to eliminate from consideration those compounds which aregenerally toxic to yeast. Initial experiments of this type have led tothe identification of compounds with potential therapeutic utility.

2. Identification of Antagonists Using Negative Selection.

Replacement of the fusl-HIS3 read-out with one of several negativeselection schemes (fusl-URA3/FOA, fusl-GAL1/galactose or deoxygalactose,Far1 sst2 or other mutations that render yeast supersensitive for growtharrest) would generate a test system in which the presence of anantagonist would result in the growth of the assay strain. Such anapproach would be applicable to high-throughput screening of compoundsas well as to the selection of antagonists from random peptide librariesexpressed in autocrine yeast. Optimization of screens of this type wouldinvolve screening the R+L+ strain at a concentration of aminotriazolewhich ablates growth of the R+L− strain (we are currently using 0.6 to0.8 mM) and counterscreening the R+L− strain at a concentration ofaminotriazole which gives an identical growth rate (we are using 0.14mM). In addition, the system could employ one of several colorometric,fluorescent or chemiluminescent readouts. Some of the genes which can befused to the fusl promoter for these alternate read-outs include lacZ(colorometric and fluorescent substrates), glucuronidase 20(colorometric and fluorescent substrates), phosphatases (e.g. PHO3,PHO5, alkaline phosphatase; colorometric and chemiuminescentsubstrates), green protein (endogenous fluorescence), horse radishperoxidase (colorometric), luciferase (chemiluminescence).

The autocrine C5a strains have further utility as follows:

3. In the Identification of Novel C5a Agonists from Random PeptideLibraries Expressed in Autocrine Yeast.

Novel peptide agonists would contribute to structure/function analysesused to guide the rational design of C5a antagonists.

4. In the Identification of Receptor Mutants.

Constitutively active, that is, ligand independent, receptors may beselected from highly mutagenized populations by growth on selectivemedia. These constitutively active receptors may have utility inpermitting the mapping of the sites of interaction between the receptorand the G-protein. Identification of those sites may be important to therational design of drugs to block that interaction. In addition,receptors could be selected for an ability to be stimulated by someagonists but not others or to be resistant to antagonist. These variantreceptors would aid in mapping sites of interaction between receptor andagonist or antagonist and would therefore contribute to rational drugdesign efforts.

5. In the Identification of Molecules that Interact with Gαi2.

Compounds or peptides which directly inhibit GDP exchange from Gαi2would have the same effect as C5a antagonists in these assays.Additional information would distinguish inhibitors of GDP exchange fromC5a antagonists. This information could be obtained through assays thatdetermine the following:

1. inhibition by test compounds of Gαi2 activation from other receptors,

2. failure of test compounds to compete with radiolabeled C5a forbinding to the C5a receptor,

3. failure of test compounds to inhibit the activation of other Gαsubunits by C5a, and

4. inhibition by test compounds of signalling from constitutively activeversions of C5a, or other, receptors.

Example 7 Construction of Xybrid Gα Genes Construction of Two Sets ofChimeric Yeast/Mammalian Gα Genes, GPA₄₁-Gα and GPA1_(Bam)-Gα

The Gα subunit of heterotrimeric G proteins must interact with both theβγ complex and the receptor. Since the domains of Gα required for eachof these interactions have not been completely defined and since ourfinal goal requires Gα proteins that communicate with a mammalianreceptor on one hand and the yeast βγ subunits on the other, we desiredto derive human-yeast chimeric Gα proteins with an optimized ability toperform both functions. From the studies reported here we determinedthat inclusion of only a small portion of the amino terminus of yeast Gαis required to couple a mammalian Gα protein to the yeast βγ subunits.It was anticipated that a further benefit to using these limitedchimeras was the preservation of the entire mammalian domain of the Gαprotein believed to be involved in receptor contact and interaction.Thus the likelihood that these chimeras would retain their ability tointeract functionally with a mammalian receptor expressed in the sameyeast cell was expected to be quite high.Plasmid Constructions.

pRS416-GPA1 (Cadus 1069). An XbaI-SacI fragment encoding the entire GPA1promotor region, coding region and approximately 250 nucleotides of 3′untranslated region was excised from 10 YCplacl 1 L-GPA1 (from S. Reed,Scripps Institute) and cloned into YEp vector pRS416 (Sikorski andHieter, Genetics 122: 19 (1989)) cut with XbaI and SacI.

Site-directed mutagenesis of GPA1 (Cadus 1075, 1121 and 1122). A 1.9 kbEcoRI fragment containing the entire GPA1 coding region and 200nucleotides from the 5′ untranslated region was cloned into EcoRI cut,phosphatase-treated pALTER-1 (Promega) and transformed byelectroporation (Biorad Gene Pulser) into DH5αF′ bacteria to yield Cadus1075. Recombinant phagemids were rescued with M13KO7 helper phage andsingle stranded recombinant DNA was extracted and purified according tothe manufacturer's specifications. A new NcoI site was introduced at theinitiator methionine of GPA1 by oligonucleotide directed mutagenesisusing the synthetic oligonucleotide:

5′ GATATATTAAGGTAGGAAACCATGGGGTGTACAGTGAG 3′ (SEQ ID NO:74).

Positive clones were selected in ampicillin and several independentclones were sequenced in both directions across the new NcoI site at +1.Two clones containing the correct sequences were retained as Cadus 1121and 1122.

Construction of a GPA1-based expression vector (Cadus 1127). The vectorused for expression of full length and hybrid mammalian Gα proteins inyeast, Cadus 1127, was constructed in the following manner. A 350nucleotide fragment spanning the 3′ untranslated region of GPA1 wasamplified with Taq polymerase (AmpliTaq; Perkin Elmer) using theoligonucleotide primers A (5′CGAGGCTCGAGGGAACGTATAATTAAAGTAGTG 3′ (SEQID NO:75) and B (5′ GCGCGGTACCAAGCTTCAATTCGAGATAATACCC 3′ (SEQ IDNO:76). The 350 nucleotide product was purified by gel electrophoresisusing GeneClean II (Biol01) and was cloned directly into the pCRIIvector by single nucleotide overlap TA cloning (InVitrogen). Recombinantclones were characterized by restriction enzyme mapping and bydideoxynucleotide sequencing. Recombinant clones contained a novel XhoIsite 5′ to the authentic GPA1 sequence and a novel KpnI site 3′ to theauthentic GPA1 sequence donated respectively by primer A and primer B.

The NotI and SacI sites in the polylinker of Cadus 1013 (pRS414) wereremoved by restriction with these enzymes followed by filling in withthe Klenow fragment of DNA polymerase I and blunt end ligation to yieldCadus 1092. The 1.4 kb PstI-EcoRI 5′ fragment of GPA1 from YCplac111-GPA1 containing the GPA1 promoter and 5′ untranslated region of GPA1 waspurified by gel electrophoresis using GeneClean (BiolO1) and cloned intoPstI-EcoRI restricted Cadus 1013 to yield Cadus 1087. The PCR amplifiedXhoI-KpnI fragment encoding the 3′ untranslated region of GPA1 wasexcised from Cadus 1089 and cloned into XhoI-KpnI restricted Cadus 1087to yield Cadus 1092. The NotI and Sac1 sites in the polylinker of Cadus1092 were removed by restriction with these enzymes, filling in with theKlenow fragment of DNA polymerase I, and blunt end ligation to yieldCadus 1110. The region of Cadus 1122 encoding the region of GPA1 fromthe EcoRI site at −200 to +120 was amplified with Vent DNA polymerase(New England Biolabs, Beverly, Mass.) with the primers

5′CCCGAATCCACCAATTTCTTTACG 3′ (SEQ ID NO:77) and

5′ GCGGCGTCGACGCGGCCGCGTAACAGT 3′ (SEQ ID NO:78).

The amplified product, bearing an EcoRI site at its 5′ end and novelSacI, NotI and SalI sites at its 3′ end was restricted with EcoRI andSalI, gel purified using GeneClean II (BiolO1), and cloned into EcoRIand SalI restricted Cadus 1110 to yield Cadus 1127. The DNA sequence ofthe vector between the EcoRI site at −200 and the KpnI site at the 3′end of the 3′ untranslated region was verified by restriction enzymemapping and dideoxynucleotide DNA sequence analysis.

PCR amplification of GPA₄₁-Gα proteins and cloning into Cadus 1127. cDNAclones encoding the human G alpha subunits Gαs, Gαi2, Gαi3, and S.cerevisiae GPA1 were amplified with Vent thermostable polymerase (NewEngland Bioloabs, Beverly, Mass.). The primer pairs used in theamplification are as follows:

GαS Primer 1: 5′CTGCTGGAGCTCCGCCTGCTGCTGCTGGGTGCTGGAG3′ (SacI 5′) (SEQID NO:79) Primer 2: 5′CTGCTGGTCGACGCGGCCGCGGGGGTTCCTTCTTAGAAGCAGC3′(SalI 3′) (SEQ ID NO:80) Primer 3: 5′GGGCTCGAGCCTTCTTAGAGCAGCTCGTAC3′(XhoI 3′) (SEQ ID NO:81) Gαi2 Primer 1:5′CTGCTGGAGCTCAAGTTGCTGCTGTTGGGTGCTGGGG3′ (SacI 5′) (SEQ ID NO:82)Primer 2: 5′CTGCTGGTCGACGCGGCCGCGCCCCTCAGAAGAGGCCGCGGTCC3′ (SalI 3′)(SEQ ID NO:83) Primer 3: 5′GGGCTCGAGCCTCAGAAGAGGCCGCAGTC3′ (XhoI 3′)(SEQ ID NO:84) Gαi3 Primer 1: 5′CTGCTGGAGCTCAAGCTGCTGCTACTCGGTGCTGGAG3′(SacI 5′) (SEQ ID NO:85) S Primer 2:5′CTGCTGGTCGACGCGGCCGCCACTAACATCCATGCTTCTCAATAAAGTC3′ (SalI 3′) (SEQ IDNO:86) Primer 3: 5′GGGCTCGAGCATGCTTCTCAATAAAGTCCAC3′ (XhoI 3′) (SEQ IDNO:87)After amplification, products were purified by gel electrophoresis usingGeneClean II (Bio101) and were cleaved with the appropriate restrictionenzymes for cloning into Cadus 1127.

The hybrid GPA₄₁-G_(α) subunits were cloned via a SacI site introducedat the desired position near the 5′ end of the amplified genes and aSalI or XhoI site introduced in the 3′ untranslated region. Ligationmixtures were electroporated into competent bacteria and plasmid DNA wasprepared from 50 cultures of ampicillin resistant bacteria.

Construction of Integrating Vectors Encoding GPA₄₁-G_(α) subunits. Thecoding region of each GPA₄₁-G_(α) hybrid was cloned into an integratingvector (pRS406=URA3 AmpR) using the BssHII sites flanking the polylinkercloning sites in this plasmid. Cadus 1011 (pRS406) was restricted withBssHII, treated with shrimp alkaline phosphatase as per themanufacturer's specifications, and the linearized vector was purified bygel electrophoresis. Inserts from each of the GPA₄₁-G_(α) hybrids wereexcised with BssHII from the parental plasmid, and subcloned into gelpurified Cadus 1011.

Construction of GPA_(BAM)-Gα Constructs. A novel BamHI site wasintroduced in frame into the GPA1 coding region by PCR amplificationusing Cadus 1179 (encoding a wildtype GPA1 allele with a novel NcoI siteat the initiator methionine) as the template, VENT polymerase, and thefollowing primers: Primer A=5′ GCATCCATCAATAATCCAG 3′ (SEQ ID NO:88) andPrimer B=5′ GAAACAATGGA -TCCACTTCTTAC 3′ (SEQ ID NO:89). The 1.1 kb PCRproduct was gel purified with GeneClean II (Biol01), restricted withNcoI and BamHI and cloned into NcoI-BamHI cut and phosphatased Cadus1122 to yield Cadus 1605. The sequence of Cadus 1605 was verified byrestriction analysis and dideoxy-sequencing of double-strandedtemplates. Recombinant GPA_(Bam)-G_(α) hybrids of Gαs, Gαi2, and Gα16were generated. Construction of Cadus 1855 encoding recombinantGPA_(Bam)-Gα 16 serves as a master example: construction of the otherhybrids followed an analogous cloning strategy. The parental plasmidCadus 1617, encoding native Gα16, was restricted with NcoI and BamHI,treated with shrimp alkaline phosphatase as per the manufacturer'sspecifications and the linearized vector was purified by gelelectrophoresis. Cadus 1605 was restricted with NcoI and BamHI and the1.1 kb fragment encoding the amino terminal 60% of GPA1 with a novelBamHI site at the 3′ end was cloned into the NcoI- and BamHI-restrictedCadus 1617. The resulting plasmid encoding the GPA_(Bam)-Gα 16 hybridwas verified by restriction analysis and assayed in tester strains roran ability to couple to yeast Gβγ and thereby suppress the gpal nullphenotype. Two additional GPA_(Bam)-Gα hybrids, GPA_(Bam)-Gαs andGPA_(Bam)-Gαi2, described in this application were prepared in ananalogous manner using Cadus1606 as the parental plasmid for theconstruction of the GPA_(Bam)-Gα i2 hybrid and Cadus 1181 as theparental plasmid for the construction of the GPA_(Bam)-Gα s hybrid.

Coupling by chimeric Gα proteins. The Gα chimeras described above weretested for the ability to couple a mammalian G protein-coupled receptorto the pheromone response pathway in yeast. The results of theseexperiments are outlined in Table 3. Results obtained using GPA1₄₁-Gαi2to couple the human C5a receptor to the pheromone response pathway inautocrine strains of yeast are disclosed in above.

Example 8 Screening for Modulators of G-Alpha Activity

Screens for modulators of Gα activity may also be performed as shown inthe following examples for illustration purposes, which are intended tobe non-limiting.

Strains CY4874 and CY4877 are isogenic but for the presence of Q205Lmutation in the cloned Gα_(i2) gene cloned into plasmid 1. StrainsCY4901 and CY4904 each have a chromosomally integrated chimeric Gαfusion comprising 41 amino acids of gpal at the N terminus of the humanGα_(i2) gene and are isogenic but for the presence of a constitutivelyactivating mutation in the C5a receptor gene of CY4901. Strain CY5058 isa gpal mutant which carries only the yeast Gβγ subunits and no Gαsubunit. This strain is a control strain to demonstrate specificity ofaction on the Gα subunit.

I. Suppression of Activation by Mutation of Gα

The Q205L mutation is a constitutively activated GTPase deficient mutantof the human Gα_(i2) gene. Antagonist compounds, chemicals or othersubstances which act on Gα_(i2) can be recognized by their action toreduce the level of activation and thus reduce the signal from thefusl-lacZ reporter gene on the second plasmid (Plasmid 2).A. GTPase Gα_(i2) Mutants

test component=gpa₄₁-Gα_(i2) (Q₂₀₅L)

control component=gpa₄₁-Gα_(i2)

As well as the CY4874 and CY4877 constructs detailed above, similarstrains with fusl-His3 or fus2-CAN-1 growth readouts may also be used.The fusl-His3 strains are preferred for screening for agonists and thefus2-CAN1 strains are preferred for antagonist screens.

test control Readout strain effect of Gα_(i2) antagonist strainfus1-HIS3 CY4868 inhibit growth of -HIS + CY4871 AT (Aminotriazole)fus1-lacZ CY4874 reduce β-gal activity CY4877 fus2-CAN1 CY4892 inducegrowth on CY4386 canavanine

In each case an antagonist should cause the test strain to behave morelike the control strain.

B. GTPase Gαs Mutants (Gα Specificity)

-   -   test component=Gα_(S)(Q₂₂₇L)    -   control component=Gα_(S)

test control Readout strain effect of Gα_(i2) antagonist strainfus1-HIS3 CY4880 none CY4883 fus1-lacZ CY4886 none CY4889 fus2-CAN1CY4895 none CY4898

In each case a non-specific antagonist would cause the test strain tobehave more like the control strain.

Additional media requirements: -TRP for Gα plasmid maintenance infusl-HIS3 and fus2-CAN1 screens and -TRP -URA for Gα and fusl-lacZplasmid maintenance in fusl-lacZ screen.

II. Suppression of Activatlon by Receptors

Constitutively Activated C5a Receptors

-   -   test component ═C5aR* (P₁₈₄L, activated C5a Receptor)    -   control component ═C5aR

The C5aR* mutation has a Leucine residue in place of the Proline residueof the wild-type at position 184 of the amino acid sequence.

test control Readout strain effect of Gα_(i2) antagonist strainfus1-HIS3 CY4029 inhibit growth of-HIS + CY2246 AT (Aminotriazole)fus1-lacZ CY4901 reduce β-gal activity CY4904 fus2-CAN1 CY4365 inducegrowth on CY4362 canavanine

In each case an antagonist should cause the test strain to behave morelike the control strain.

Additional media requirements: -LEU for receptor plasmid maintenance infusl-HIS3 and fus2-CAN1 screens and -LEU-URA for receptor and ftisl-lacZplasmid maintenance in fus1-lacZ screen, non-buffered yeast media (pH5.5).

Example 9 Identification of a Surrogate Ligand Using Expression of aRandom Peptide Library in Yeast Expressing an Orphan Mammalian Receptor

FPRL-1 (formyl peptide receptor-like 1) is a structural homolog of theformyl peptide receptor (FPR). FPR is a G protein-coupled receptor,expressed on neutrophils and phagocytic cells, that is stimulated byN-formyl peptides of bacterial origin. Specific binding of the naturalligand, f-Met-Leu-Phe, stimulates transduction of a signal to mobilizecalcium, resulting in cellular changes including chemotaxis and therelease of granule contents. Low stringency hybridization of HL60 cDNAlibraries with an FPR cDNA probe permitted the identification of therelated receptor, FPRL-1 (Murphy et al. supra; Ye et al. supra). TheFPRL-1 cDNA encodes a 351 amino acid protein with 69% sequence homologyto FPR (Murphy et al. supra) FPR and FPRL-1 were found to co-localize tohuman chromosome 19 and to have a tissue expression pattern identical tothat of FPR, i.e., expression is restricted to cells of myeloid origin(Murphy et al. supra). Ye et al. (supra) demonstrated weak binding off-Met-Leu-Phe (uM concentrations) to fibroblasts transfected with FPRL-1cDNA. In contrast, Murphy et al. (supra) could not detect binding ofN-formyl peptides to Xenopus oocytes transfected with FPRL-1 cDNA.FPRL-1 appears to be an orphan receptor whose specific ligand differsfrom the formyl peptide ligands to which FPR responds.

In this example experiments detailing the following will be described:(1) establishment of a strain of yeast designed to express the humanorphan G protein-coupled receptor FPRL-1; (2) expression of a randompeptide library in the aforementioned strain of yeast; and (3)activation of the endogenous yeast pheromone pathway upon stimulation ofthe FPRL-1 receptor by a peptide encoded by a random library expressedwithin the same strain of yeast.

Preparation of FPRL-1 Yeast Expression Vector

A plasmid, pFPRL1-L31, containing a 2.6 kb EcoRI-Xho1 fragment encodingthe FPRL-1 cDNA in the BluescriptIISK+ vector was obtained from PhilipMurphy (NIH). The sequence encoding FPRL1 was amplified by thepolymerase chain reaction using VENT polymerase (New England Biolabs,Inc., Beverly, Mass.) through 20 cycles and the followingoligonucleotide primers:

#1 5′ GGCGCCCGGTCTCCCATGGAAACCAACTTCTCCACT (SEQ ID NO:90) #2 5′GGCGCCCGGTCTCCGATCCCATTGCCTGTAACTCAGTCTC (SEQ ID NO:91)The PCR product was purified, restricted with BsaI and cloned into Cadus1651 (plPBX-1), a PGK promoter-driven expression vector, using NcoI andBamHI sites, to yield CADUS 2311. The sequence of the entire insert wasdetermined and found to be identical to the FPRL-1 sequence deposited inGenBank (accession number M84562).Preparation of Random Oligonucleotides

Library-Recycling Protocol to Identify a Surrogate Ligand

The yeast strain CY1141 (MATalpha far1*1441 tbt1-1 fusl-HIS3 can lste14::Trpl::LYS2 ste3*1156 gpal(41)-Galphai2 lys2 ura3 leu2 trp1 his3)was used in the experiments that follow. CY1141 contains a pheromoneinducible HIS3 gene, fusl-HIS3 integrated at the FUS1 locus and a hybridgene encoding the first 41 amino acids of GPA1 (yeast G alpha) fused tosequence encoding human G alphai2 (lacking codons encoding theN-terminal 33 amino acids) replacing GPA1 at its chromosomal locus. Theyeast STE14 gene is disrupted to lower the basal level of signalingthrough the pheromone response pathway. The yeast a-factor receptorgene, STE3, is deleted. CY1141 was transformed with Cadus 2311 to yieldCY6571, a strain expressing the human orphan receptor, FPRL-1.

CY6571 exhibited LIRMA (ligand independent receptor mediatedactivation), that is, activation of the yeast pheromone pathway in theabsence of ligand. It was determined that the yeast growth on selectivemedia that resulted from LIRMA was eliminated by the additional of 2.5millimolar concentrations of 3-aminotriazole (AT). AT is an inhibitor ofthe HIS3 gene product that serves to reduce background growth.Therefore, selection protocols aimed at the identification of surrogateligands for the FPRL-1 receptor were carried out at this concentrationof AT.

CY6571 was inoculated to 10 mls of standard synthetic media (SD) lackingleucine (-Leu) and incubated overnight at 30° C. The 10 ml overnightculture was used to inoculate 50 mls of YEPD; this culture was incubatedat 30° C. for 4.5–5 hours at which time the cells were harvested andprepared for transformation with DNA encoding a random peptide library[alpha-NNK (6.24.94)] encoding tridecapeptides of random sequence, byelectroporation. Post electroporation (in 0.2 cm cuvettes, 0.25 μF, 200Ω, 1.5 kV) the cells were immediately diluted in 1 ml ice-cold 1Msorbitol and 100 μL aliquots were placed onto 10 synthetic media plates(pH6.8) lacking leucine and uracil (-Leu-Ura). The plates were incubatedat 30° C. for 2–4 days at which time two replicas of each originaltransformation plate were made to synthetic media (pH6.8) lackingleucine, uracil and histidine and supplemented with 2.5 mMAT(-Leu-Ura-His+2.5 mM AT). The replicas were incubated at 30° C. for3–5 days. Post incubation the colonies present on the replica sets oftwo were scraped from the plates into a total of 10 mls of H₂O (5 mlseach plate). The OD₆₀₀ of each cell suspension was determined and crudeplasmid isolations were done on 8–16 OD units of cells for each pool. Atotal of eight pools resulted, due to lower numbers of yeast coloniespresent in four sets of plates. The pellets obtained from these crudeplasmid isolations (the so called “smash and grab” technique, Methods inYeast Genetics—A Laboratory Manual, 1990, M. D. Rose, F. Winston and P.Heiler. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),were resuspended in 40 μL of 10 mM Tris, 1 mM EDTA, pH8.0 and 1 μL wasused to transform E. coli by electroporation (0.1 cm cuvettes, 0.25 μF,200 Ω, 1.8 kV). Post electroporation the cells were immediately dilutedinto 1 ml 2XYT media and incubated, with shaking, at 37° C. for 30minutes after which time the cells were used to inoculate 50 mls of 2xYTsupplemented with 100 ug/ml ampicillin. The 10 resulting cultures wereincubated at 37° C. overnight. Plasmid DNA was isolated from each ofthese bacteria cultures using Qiagen columns (Qiagen, Inc., Chatsworth,Calif.)). Each plasmid DNA pellet was resuspended in 50 μL Tris 10 mM,EDTA 1 mM, pH 8.0.

Strain CY6571 was transformed with 1 μL of each plasmid pool byelectroporation. Post electroporation the cells were diluted into 400 μL1M sorbitol. From each electroporated cell suspension, 1 μL and 400 μLof cells were plated on -Leu-Ura synthetic media, pH6.8 to yield “lowdensity” and “high density” platings. The plates were incubated at 30°C. for 3 days, at which time replicas of both the low and high densityplates were made to -Leu-Ura-His+2.5 mM AT. For those cases whereenrichment for a plasmid capable of conferring a His+ phenotype hadoccurred, this would be reflected by an amplified number of His+colonies on both the low and high density plates visible at days 2–3,although the amplification would be most obvious on the plates that hadreceived a high density of cells. In the FPRL-1 experiment ⅛ poolsshowed amplification of His+ colonies. The cells were scraped from thisplate into 5 mls of H₂O, the OD₆₀₀ of the cell suspension was determinedand a crude plasmid isolation was done on 15 OD units of yeast cells.The pellet obtained was resuspended in 40 μL 10 mM Tris, 1 mM EDTA,pH8.0 and 1 μL was used to transform E. coli. Plasmid DNA was isolatedby miniprep from 3 ml 2XYT cultures of single bacterial coloniesresulting from this transformation. 10 DNA pellets (A1 through A10)deriving from individual bacterial colonies were resuspended in 20 μL 10mM Tris 1 mM EDTA, pH8.0 and used to transform CY6571 (containing theFPRL-1 expression vector) and CY6263 (CY1141 containing a controlexpression vector lacking any receptor sequence) by electroporation.Cadus 1625, a control vector lacking sequences encoding a peptide, wasincluded and used to transform both the receptor+ and receptor− strainsof yeast. Transformants were first selected on -Leu-Ura, pH6.8 thenthree yeast transformants of each type (from 11 CY6571 transformationsand 11 CY6263 transformations) were patched to -Leu-Ura, pH6.8 to expandthe colonies. Once expanded, streaks of the transformants were made on-Leu-Ura-His+2.5 mM AT to test for growth in the absence of histidine.All plasmids except the one denoted A2 conferred a growth advantage onmedia lacking histidine to yeast bearing the FPRL-1-encoding plasmid butnot to yeast lacking the receptor plasmid. The peptide sequence found tobe encoded by plasmids A1 and A3–A10 is:SerLeuLeuTrpLeuThrCysArgProTrpGluAlaMet, and is encoded by thenucleotide sequence 5′-TCT CTG CTT TGG CTG ACT TGT CGG CCT TGG GAG GCGATG-3′ (SEQ ID NO:92).

Activation of the Pheromone Response Pathway in Yeast Expressing theFPRL-1 Receptor and Peptide Agonist.

For verification of pheromone pathway activation and quantification ofthe stimulation, the activity of the fusl promoter was determinedcolorimetrically using a fus1-lacZ fusion in a parallel set of teststrains. CY 1141, described above, was used as the recipient strain forthese experiments. Transformants contained CADUS 1584 (pRS424-fusl-lacZ)in addition to receptor (R^(+/−)) and ligand (L^(+/−)) plasmids. Fourstrains (bearing the identical plasmids) were grown overnight in minimalmedia lacking leucine, uracil, and tryptophan, pH8.6. The overnightcultures were used to inoculate -Leu -Ura -Trp pH6.8 media and these newcultures were grown for approximately 4.5–5 hours to an OD₆₀₀ of lessthan 0.4. Assay of β-galactosidase activity (Guarente 1983) in cellsfrom these cultures yielded the following results:

CY1141/CADUS 2311/peptide A1/CADUS 1584 R⁺L⁺ 28 units CY1141/CADUS2311/CADUS 1625/CADUS 1584 R⁺L⁻ 3 units CY1141/CADUS 1289/peptideA1/CADUS 1584 R⁻L⁺ 3.5 units CY1141/CADUS 1289/CADUS 1625/CADUS 1584R⁻L⁻ 3.9 unitsThe presence of receptor and peptide-encoding plasmids resulted in anaverage 8-fold stimulation over background levels of β-galactosidase.

Example 10 Identification of Surrogate Ligands Using Expression of aRandom Peptide Library in Yeast Expressing the Orphan MammalianReceptor, MDR-15

In a similar manner a plasmid encoding the monocyte derived receptormonocyte-derived receptor 15 (MDR15; Barella et al. (1995) Biochem. J.309:773–9) was used to construct a yeast strain (CY6573) expressing thisreceptor. This receptor is an alternative spliced form of the Burkitt'slymphoma receptor 1 (BLR1) encoded by a human Burkitt's lymphoma cDNA(Dobner et al. (1992) Eur. J. Immunol. 22, 2795–2799). Strain CY6573 wastransformed in a similar manner with the NNK13 library, and, followingselection on ten-Leu-Ura (4.4×10⁵ colonies per plate), replica plated to-Leu-Ura-His+ 1 mM AT plates. Upon reisolation of plasmid pools andre-transformation into strain CY6573; eight of ten pools showedsignicantly enriched colony formation on -Leu-Ura-His+ 1 mM AT plates.Eight unique plasmids derived from these pools when retransformed intoCY6573 conferred growth on -Leu-Ura-His+ 1 mM AT plates. One of theseplasmids failed to confer growth in a yeast strain lacking the MDR15receptor.

Example 11 Identification of a Ligand Using Expression of a RandomPeptide Library in Yeast Expressing the Human Thrombin Receptor

The receptor for thrombin, a G protein-coupled receptor, is present onnumerous cell types including platelets, vascular smooth muscle,fibroblasts and on a subset of cells that function in immunity.Thrombin, a serine protease, binds to and cleaves the receptor moleculeat residue 41, generating a new receptor N-terminus. The post-cleavageN-terminal residues then act as a “tethered ligand” to activate thereceptor molecule (Vu et al. 1994). In platelets, signaling through thethrombin receptor has been shown to result in numerous effects includingstimulation of phospholipase C, mobilization of intracellular Ca²⁺ andinhibition of adenylyl cyclase.

In this example experiments that detail the following will be described(1) establishment of a strain of yeast designed to express the human Gprotein-coupled receptor for thrombin; (2) expression of a randompeptide library in the afore-mentioned strain of yeast and (3)activation of the endogenous yeast pheromone pathway upon stimulation ofthe thrombin receptor by peptides encoded by a random library expressedwithin the same strain of yeast.

Preparation of a Yeast Expression Vector for a Mammalian ThrombinReceptor

The human thrombin receptor was amplified by PCR from pcDNA3:Hu-Thr9b-5′(Bristol Myers Squibb) using the following oligonucleotides:

5′ GGGCCATGGGGCCGCGGCGGTTG 3′ (SEQ ID NO: 93) 5′CCCGGATCCTAAGTTAACAGCTTTTTGTATAT 3′ (SEQ ID NO: 94)The amplified product was purified by gel electrophoresis, restrictedwith NcoI and BamHI and ligated to NcoI and BamHI-cut CADUS 1871, a PGKpromoter-driven expression vector, to yield CADUS 2260. Cloning intoCADUS 1871 introduces a novel stop codon preceded by the tripletGlySerVal after the authentic carboxy terminal codon of the humanthrombin receptor (threonine). In addition, an invertase signal sequenceis fused to the authentic amino terminus of the receptor.

CY7467 exhibited LIRMA (ligand independent receptor mediatedactivation), that is, activation of the yeast pheromone pathway in theabsence of ligand. It was determined that the yeast growth on selectivemedia that resulted from LIRMA was eliminated by the addition of 2.5millimolar concentrations of 3-aminotriazole (AT). AT is an inhibitor ofthe HIS3 gene product that serves to reduce background growth.Therefore, selection protocols aimed at the identification of novelpeptide ligands for the human thrombin receptor were carried out at thisconcentration of AT.

Preparation of Random Oligonucleotide Library

As described above.

Recycling Protocol to Identify a Surrogate Ligand

The yeast strain CY1141 (MATalpha far1*1442 tbt1-1 fusl-HIS3 can1stel4::Trp1::LYS2 ste3*1156 gpal(41)-Galphai2 lys2 ura3 leu2 trp1 his3)was transformed with CADUS 2260 to yield strain CY7467, expressing thehuman thrombin receptor. CY7467 was inoculated to 10 mls of standardsynthetic media (SD) lacking leucine (-Leu) and incubated overnight at30 C. The 10 ml overnight culture was used to inoculate 50 mls of YEPDmedia; this culture was incubated at 30 C for 4.5–5 hours at which timethe cells were harvested and prepared for transformation with DNAencoding a random peptide library [alpha-NNK (6.24.94)] byelectroporation. Post electroporation (in 0.2 cm cuvettes, 0.25 mF, 200W, 1.5 kV) the cells were immediately diluted in 1 ml ice-cold 1Msorbitol and 100 mL aliquots were plated onto 10 synthetic media plates(pH6.8) lacking leucine and uracil (-Leu-Ura). The plates were incubatedat 30 C for 2–4 days at which time two replicas of each originaltransformation plate were made to synthetic media (pH6.8) lackingleucine, uracil and histidine and supplemented with 2.5 mMAT(-Leu-Ura-His+ 2.5 mM AT). The replicas were incubated at 30 C for 3–5days. Post incubation the colonies present on the replica sets of twowere scraped from the plates into a total of 10 mls of H₂O (5 mls eachplate). The OD₆₀₀ of each cell suspension was determined and crudeplasmid isolations were done on 8–16 OD units of cells for each pool. Atotal of ten pools resulted. The pellets obtained from these crudeplasmid isolations were resuspended in 40 mL of 10 mM Tris, 1 mM EDTA,pH8.0 and 1 mL was used to transform E. coli by electroporation (0.1 cmcuvettes, 0.25 mF, 200 W, 1.8 kV). Post electroporation the cells wereimmediately diluted into 1 ml 2XYT media and incubated, with shaking, at37 C for 30 minutes after which time the cells were used to inoculate 50mls of 2xYT supplemented with 100 ug/ml ampicillin. The 10 resultingcultures were incubated at 37 C overnight. Plasmid DNA was isolated fromeach of these bacterial cultures using Qiagen columns (Qiagen, Inc.,Chatsworth, Calif.). Each plasmid DNA pellet was resuspended in 50 mLTris 10 mM, EDTA 1 mM, pH 8.0.

Strain CY7467 was transformed with 1 mL of each plasmid pool byelectroporation. Post electroporation the cells were diluted into 400 mL1M sorbitol. From each electroporated cell suspension, 1 mL and 400 mLof cells were plated on -Leu-Ura synthetic media, pH6.8 to yield “lowdensity” and “high density” platings. The plates were incubated at 30 Cfor 3 days, at which time replicas of both the low and high densityplates were made to -Leu-Ura-His+ 2.5 mM AT. For those cases whereenrichment for a plasmid capable of conferring a His+ phenotype hadoccurred, this would be reflected by an amplified number of His+colonies on both the low and high density plates visible at days 2–3,although the amplification would be most obvious on the plates that hadreceived a high density of cells. In this experiment 3/10 pools showedamplification of His+ colonies. The cells from each of these plates werescraped into 5 mls of H₂O, the OD₆₀₀ of the cell suspensions weredetermined and crude plasmid isolations were done on 8–160D units ofyeast cells. The pellets obtained were resuspended in 40 mL 10 mM Tris,1 mM EDTA, pH8.0 and 1 mL was used to transform E. coli. Plasmid DNA wasisolated by miniprep from 3 ml 2XYT cultures of single bacterialcolonies resulting from these transformations (three bacterial coloniesfor each DNA pool were processed in this way). DNAs deriving from threeindividual bacterial colonies per pool were resuspended in 20 mL 10 mMTris 1 mM EDTA, pH8.0. The three DNAs derived per pool were sequencedand found to encode identical peptides. Thus three differing DNAsequences were derived, one representing each amplified pool. Oneplasmid representing each of the three original amplified pools was usedto transform CY7467 (containing the thrombin receptor expression vector)and CY6263 (CY1141 containing a control expression vector lacking anyreceptor sequence) by electroporation. CADUS 1625, a control vectorlacking sequences encoding a peptide was included and used to transformboth the receptor+ and receptor− strains of yeast. CADUS 1651, a controlvector lacking sequences encoding a receptor included and used totransform both the ligand+ and ligand-strains of yeast. Transformantswere first selected on -Leu-Ura, pH6.8, then two yeast transformants ofeach type were patched to -Leu-Ura, pH6.8 to expand the colonies. Onceexpanded, streaks of the transformants were made on -Leu-Ura-His+ 2.5 mMAT to test for growth in the absence of histidine. One of the threeplasmids tested conferred a growth advantage on media lacking histidineto yeast bearing the thrombin-encoding plasmid but not to yeast lackingthe receptor plasmid. The peptide sequence encoded by this plasmid is:Val-Cys-Pro-Ala-Arg-Tyr-Val-Leu-Pro-Gly-Pro-Val-Leu (SEQ ID NO:96) andwas encoded by the nucleotide sequence GTT TGT CCT GCG CGT TAT GTG CTGCCT GGG CCT GTT TTG (SEQ ID NO:95).

1. A mixture of recombinant yeast cells, each cell of which comprises:(i) a recombinant gene encoding a heterologous orphan G protein-coupledreceptor wherein said receptor is expressed on the cell membrane of saidcell such that signal transduction activity is modulated by interactionwith an extracellular signal; and (ii) a recombinant gene encoding aheterologous test polypeptide, wherein the test polypeptide istransported to a location allowing interaction with the receptorexpressed on the cell membrane, wherein collectively the mixture ofcells expresses a library of said test polypeptides, and modulation ofthe signal transduction activity of the receptor by a heterologous testpolypeptides within the library provides a detectable signal.
 2. Amixture of recombinant yeast cells, each cell of which comprises: (i) aheterologous orphan G protein-coupled receptor wherein said receptor isexpressed on the cell membrane of said cell such that signaltransduction activity is modulated by interaction with an extracellularsignal; (ii) a recombinant gene encoding a heterologous testpolypeptide, receptor, wherein the test polypeptide is transported to alocation allowing interaction with the receptor expressed on the cellmembrane; and (iii) a reporter gene construct containing a reporter genein operative linkage with one or more transcriptional regulatoryelements responsive to the signal transduction activity of the receptor,wherein collectively the mixture of cells expresses a library of testpolypeptides.
 3. A mixture of recombinant yeast cells, each cell ofwhich comprises: (i) an orphan G protein-coupled receptor wherein saidreceptor is expressed on the cell membrane of said cell such that signaltransduction activity is modulated by interaction with an extracellularsignal; (ii) a recombinant gene encoding a heterologous test polypeptideand including a signal sequence for secretion, wherein the testpolypeptide is transported to a location allowing interaction with thereceptor expressed on the cell membrane; and (iii) a reporter geneconstruct containing a reporter gene in operative linkage with one ormore transcriptional regulatory elements responsive to the signaltransduction activity of the receptor, wherein collectively the mixtureof cells expresses a library of test polypeptides.
 4. A mixture ofrecombinant yeast cells, each cell of which comprises: (i) an orphan Gprotein-coupled receptor wherein said receptor is expressed on the cellmembrane of said cell such that signal transduction activity ismodulated by interaction with an extracellular signal; and (ii) arecombinant gene encoding a heterologous test polypeptide and includinga signal sequence for secretion, wherein the test polypeptide istransported to a location allowing interaction with the receptorexpressed on the cell membrane, wherein collectively the mixture ofcells expresses a library of test polypeptides, and modulation of thesignal transduction activity of the receptor by a test polypeptidewithin the library provides a detectable signal.
 5. The mixture of claim4, wherein each cell further comprises a reporter gene constructcontaining a reporter gene in operative linkage with one or moretranscriptional regulatory elements responsive to the signaltransduction activity of the receptor, expression of the reporter geneproviding the detectable signal.
 6. The mixture of claim 4, wherein thereporter gene encodes a gene product that gives rise to a fluorescencedetectable signal.
 7. The mixture of claim 5, wherein the reporter geneencodes a beta-galactosidase gene product.
 8. The mixture of claim 4,wherein each cell further comprises a heterologous gene constructencoding the receptor.
 9. The mixture of claim 4, wherein the variegatedpopulation of test polypeptides includes at least 10³ different testpolypeptides.
 10. A recombinant yeast cell, comprising: (i) arecombinant gene encoding a heterologous G protein-coupled receptorprotein wherein said receptor is expressed on the cell membrane of saidcell such that signal transduction activity is modulated by anextracellular signal; (ii) a recombinant gene encoding a heterologoustest polypeptide, wherein the test polypeptide is transported to alocation allowing interaction with the receptor expressed on the cellmembrane; and (iii) a reporter gene construct containing a reporter genein operative linkage with one or more transcriptional regulatoryelements responsive to the signal transduction activity or the receptor.11. The recombinant cell of claim 10, wherein the reporter gene encodesa fluorescence gene product that gives rise to a fluorescence detectablesignal.
 12. The recombinant cell of claim 10, which yeast cell is aSaccharomyces cell.
 13. The recombinant cell of claim 10, which yeastcell is a Schizosaccharomyces cell.
 14. A mixture of recombinant yeastcells, each cell of which comprises: (i) a recombinant gene encoding aheterologous orphan G protein-coupled receptor wherein said receptor isexpressed on the cell membrane of said cell such that signaltransduction activity is modulated by interaction with an extracellularsignal; (ii) a recombinant gene encoding a heterologous test polypeptideand including a signal sequence for secretion, wherein the testpolypeptide is transported to a location allowing interaction with thereceptor expressed on the cell membrane; and (iii) a reporter geneconstruct containing a reporter gene in operative linkage with one ormore transcriptional regulatory elements responsive to the signaltransduction activity of the receptor, wherein collectively the mixtureof cells expresses a library of test polypeptides.
 15. The mixture ofclaim 14, which yeast cell is a Saccharomyces cell.
 16. The mixture ofclaim 14, which yeast cell is a Schizosaccharornyces cell.
 17. Themixture of claim 14, wherein the variegated population of testpolypeptides includes at least 10³ different test polypeptides.
 18. Themixture of any of claims 1, 2, 3, 4 or 14, wherein the G protein-coupledreceptor is selected from the group consisting of a chemoattractantpeptide receptor, a neuropeptide receptor, a light receptor, aneurotransmitter receptor, a cyclic AMP receptor, and a polypeptidehormone receptor.
 19. The recombinant yeast cell of claim 10, whereinthe G protein-coupled receptor is selected from the group consisting ofa chemoattractant peptide receptor, a neuropeptide receptor, a lightreceptor, a neurotransmitter receptor, a cyclic AMP receptor, and apolypeptide hormone receptor.